Method for producing a novel purified Haemophilus influenzae protein

ABSTRACT

Disclosed herein are immunogenic polysaccharide-H. influenzae adhesin protein conjugates, a purified H. influenzae adhesin protein and related proteins and polypeptides, DNA useful for producing the proteins, synthetic polyribosylribotol phosphate (PRP) oligosaccharides and intermediates useful for their synthesis, and methods of making and using these materials. The conjugates comprise a PRP fragment, preferably a synthetic oligosaccharide, coupled to an H. influenzae adhesin protein. The invention further comprises purified H. influenzae adhesin proteins and novel PRP oligosaccharides. The invention also comprises methods of producing these materials and using them in a vaccine to protect humans and other mammals against H. influenzae infection.

BACKGROUND OF THE INVENTION

This is a divisional application of application Ser. No. 07/903,079,filed on Jun. 22, 1992, which is a continuation-in-part application ofapplication Ser. No. 07/810,966 filed on Dec. 20, 1991, which is acontinuation-in-part application of application Ser. No. 07/631,698,filed on Dec. 21, 1990, all of which are incorporated herein byreference in their entirety.

This invention relates generally to vaccines against Haemophilusinfluenzae. In particular, it relates to a conjugate vaccine in which asynthetic oligosaccharide corresponding to a fragment of thepolysaccharide capsule of H. influenzae type b has been coupled to an H.influenzae adhesin protein. The vaccine may be used against bothinvasive and non-invasive H. influenzae infection of humans,particularly very young infants, and other mammals.

H. influenzae (Hi) are divided into two groups, those strains thatpossess a polysaccharide capsule and those that do not. The encapsulatedstrains are typed by a serological reaction of a capsule with referenceantisera. Types a-f have been identified. The non-encapsulated strains,which fail to react with any of the reference antisera, are known asnon-typeable.

Hi are a significant health problem worldwide. The type b strain (Hib)is the most virulent of the Hi strains, causing meningitis, acuteepiglottitis, and other life-threatening infections in children fiveyears old and younger. The mortality rate from type b meningitis isabout 5%, even with the best modern antibiotic treatments, andneurological sequelae are observed in as many as 25-35% of thesurvivors. In fact, bacterial meningitis caused by type b strains hasbeen identified as the leading cause of acquired mental retardation inthe United States. Thus, World Health Organization has made thedevelopment of an effective vaccine against Hib a priority.

Non-typeable Hi also cause various diseases, including pneumonia,bacteremia, meningitis, postpartum sepsis, bronchitis, sinusitis,conjunctivitis, and otitis media. The non-typeable Hi cause about 20-40%of all otitis media in children and young adults. Current therapy forchronic or repeated occurrences of otitis media generally involvesantibiotic administration. Children may experience multiple infectionsbecause infection does not confer a lasting immunity.

A great deal of time, money, and effort has been spent trying to find atruly effective vaccine to H. influenzae. The overwhelming focus hasbeen on developing a vaccine for Hib because of its serious threat tovery young children. Unfortunately, the approved type b polysaccharidevaccines are not effective for children under 18 months of age, which isthe group most threatened by Hib.

It has been known for many years that antibodies directed against thetype b capsule will protect individuals against invasive Hib infection,including meningitis. In a randomized, double-blind clinical trial inFinland, a type b polysaccharide vaccine was found to be 90% effectivein presenting disease in children immunized between 24 and 72 months ofage. However, the vaccine conferred no protective immunity in childrenyounger than 18 months and provided only limited immunity in childrenaged 18-23 months. Peltola, et al., N. Engl. J. Med., 310:1561-1566(1984). The type b polysaccharide elicits a T-cell-independent immuneresponse, which probably accounts for the low immunogenicity in youngchildren.

Based on these data, three type b polysaccharide vaccines were licensedin the United States in 1985 and were recommended for use in childrenaged 24-60 months. These vaccines obviously have a major problem. Theydo not adequately protect children under 24 months of age, the groupmost succeptible to H. influenzae disease.

There are other problems relating to the fact that the polysaccharide isobtained from natural sources. Although purified, the polysaccharidefragments are of various lengths and, therefore, not as wellcharacterized as desirable. This creates problems with respect toreproducibility and variable potency. Also, since naturally occurringpolysaccharide must be isolated from a pathogen, safety concerns must beaddressed with respect to both manufacture and use of the vaccine.

Attempts have been made to make the polysaccharide into a betterimmunogen. The polysaccharide or fragments thereof have been covalentlycoupled with various immunogenic proteins, such as diphtheria or tetanustoxoids. See, for example, U.S. Pat. No. 4,673,574 issued Jun. 16, 1987to Anderson, U.S. Pat. No. 4,808,700 issued Feb. 28, 1989 to Anderson,et al., European Patent Office Publication No. 0 245 045 dated Nov. 11,1987, and European Patent Office Publication No. 0 098 581 dated Jan.18, 1984, all of which are incorporated herein by reference.

Several of the conjugate vaccines have been shown to be safe and moreimmunogenic than the conventional polysaccharide vaccines in children,particularly infants. The data suggest that the conjugate vaccines arefunctioning as T-cell dependent antigens. A T-cell-dependent responseprovides for a better overall immune response in a patient. One of theconjugate vaccines has been approved in the United States for children15-18 months of age. Two of the conjugate vaccines have been licensed inthe United States for infants as young as two months old.

The vaccines currently available to the medical practitioner haveseveral major limitations. First, they do not protect against other Hiinfections besides Hib. The polysaccharide is not found in non-typeableH. influenzae; therefore, antibodies to it are non-protective againstthese strains. Second, they raise problems with respect toreproducibility, potency, and safety.

There are several avenues of on-going research on ways to overcome theselimitations. One approach has been to develop procedures for Hibpolysaccharide synthesis. The Hib capsule consists of a linearhomopolymer of alternating molecules of ribose and ribitol joined by aphosphodiester linkage represented by the following formula: ##STR1##The polymer is known as polyribosylribitol phosphate and abbreviatedPRP.

The PRP obtained from natural sources is crude degraded polysaccharide.It varies in molecular weight between 200 KD and 200,000 KD.

A few groups have been able to synthesize small PRP oligosaccharides.For example, European Patent Office Publication 0 320 942 dated Jun. 21,1989, incorporated herein by reference, discloses the synthesis ofsynthetic PRP oligosaccharides of 2-20 units and their covalentattachment to immunogenic proteins, specifically tetanus or diptheriatoxins or toxoids. The oligosaccharides are linked to the proteinsthrough a spacer. A phosphite triester synthetic procedure was used forthe oligomerization. European Patent Office Publication 0 276 516 datedAug. 3, 1988, incorporated herein by reference, also discloses syntheticPRP oligosaccharides 2-20 monomers in length, their conjugation tocarrier proteins, and the use of the conjugates as vaccines against Hib.The oligosaccharides are prepared using the phosphotriester syntheticprocedure for oligomerization. Both of these involved solution-typesynthetic techniques for the preparation of the PRP oligosaccharides.

Elie, et al., Recl. Trav. Chim. Pays-Bas, 108:219-223 (1989),incorporated herein by reference, discloses the solid-phase synthesis ofa PRP hexamer. The units were coupled using a phosphite triester methodand controlled-pore glass as the solid support.

The use of synthetic PRP fragments should provide several advantagesover the PRP obtained from natural sources. Synthetic PRP is chemicallywell-defined and characterized. It would be of superior quality and lessprone to produce side effects in humans. Its use would also obviateproblems relating to reproducibility, potency, and safety associatedwith PRP obtained from natural sources. In addition, while the naturallyoccurring PRP is generally cross-linked to the protein carrier at randompoints along its chain, synthetic PRP can be conjugated through a singlepoint, which creates less undesired epitopes.

This research promises improvements to existing vaccines, but there arestill drawbacks. First, the PRP synthesis is complicated and relativelyinefficient. Thus, there is a need for improved synthesis procedures.Second, these improvements will be limited to vaccines against Hib.

Another approach has been to focus on the protein. There are someavailable data suggesting that the protein and the carbohydrate parts ofthe conjugate vaccines act as independent immunogens. Therefore, thechoice of the protein component becomes important in seeking to enhanceimmunogenicity. It would be more desirable to have an immunogenicprotein or polypeptide derived from H. influenzae as the proteincomponent rather than a "nonsense" protein.

At least one group has conjugated an Hib outer membrane protein to PRPfragments. See European Patent Office Publication No. 0 338 265, datedOct. 25, 1989, incorporated herein by reference. This applicationdiscloses 38 and 40 KD outer membrane proteins of Hib and theirisolation and purification. The two proteins are quite similar. They areknown as protein 2 (P2) or protein b/c because they occur as a doublet.The molecular weight depends upon the strain from which they areobtained. They are cross-reactive, have very similar amino acidcompositions, and have the same amino and carboxy terminal sequences.The proteins are coupled to PRP fragments by reductive amination. ThePRP fragments are obtained from naturally occurring PRP using standardtechniques. The application states that the carrier proteins themselvesmay confer immunity.

This approach also suffers from certain limitations. The outer membraneproteins may vary among Hib types or serotypes within a particular type.Granoff, et al., in S. H. Sell and P. F. Wright (ed.), HaemophilusInfluenzae: Epidemiology, Immunology, and Prevention, (New York:Elsevier Biomedical (1982)). Therefore, a vaccine based upon aparticular outer membrane protein may not be effective against thebroader spectrum of pathogenic H. influenzae bacteria and may not evenbe effective against all strains of Hib.

Others have focused on Hi proteins and peptides alone as vaccinecandidates. For example, see PCT Publication No. WO 90/02557, publishedMarch 22, 1990, incorporated herein by reference. This applicationdiscloses two antigenically related Hi outer membrane proteins with amolecular weight of about 16 KD. It further discloses related fusionproteins and peptide fragments of the outer membrane proteins, methodsof purifying the proteins, and methods of making them by geneticengineering. All of these are claimed to be useful as immunogens invaccines. Such vaccines will also have the drawbacks mentionedimmediately above.

Clearly, there is a pressing need for a safe vaccine that is effectiveagainst both invasive and non-invasive H. influenzae, particularly ininfants 2-6 months old. This ideal vaccine would also be effectiveagainst a wide variety of strains within each of the two categories byeliciting antibodies against a determinant found on the surface of mostor all strains of H. influenzae.

The present invention overcomes the limitations of the existingtechnology and meets that need. It provides a novel synthetic PRPconjugated to newly isolated and purified H. influenzae adhesinproteins.

The ability to use an adhesin protein in a vaccine against H. influenzaeis extremely desirable. Because of the way they function, adhesinproteins are believed to be highly conserved among strains of aparticular type of bacteria. This is because they are the proteinmolecules that mediate attachment by bonding bacteria to host cells, theinitial step in the infection process. Thus, the adhesins would beexpected to be present in all strains (both encapsulated andunencapsulated) of Haemophilus. Therefore, the present vaccine would beeffective against a broad array of types and strains of Hi. In addition,vaccines based upon adhesin proteins should be more effective than thosebased upon other outer membrane proteins, even for those bacterialstrains from which the outer membrane proteins are derived. Antibodiesto the adhesin protein would prevent adherence of the bacteria to thetissue of the host animal. Adherence is the initial step in Hiinfection. Stopping the infection at this point would be the bestapproach possible.

The novel PRP of the invention also has advantages over the existingtechnology. It is better defined and characterized, and it is ofsuperior quality when compared to PRP obtained from natural sources.Also, it has been more efficiently produced than the synthetic PRPdescribed above.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an immunogenicoligosaccharide-H. influenzae adhesin protein conjugate and a method formaking it.

Another object of the invention is to provide a vaccine for protecting amammal against H. influenzae.

Yet another object of the invention is to provide a method of inducingan immune response to H. influenzae in a mammal.

A further object of the invention to provide purified H. influenzaeadhesin proteins.

A still further object of the invention is to provide a purifiedpolypeptide capable of eliciting an antigenic response to H. influenzaein an animal host.

Yet another object of the invention is to provide methods for producingpurified H. influenzae adhesin proteins.

A further object of the invention is to provide DNA coding for theadhesin proteins and derived polypeptides, vectors containing the DNA,microorganisms transformed by such DNA and vectors, and methods forpreparing such materials.

A still further object of the invention is to provide a composition ofmatter consisting essentially of synthetic PRP oligosaccharides havingthe same number of monomeric units and a method of preparing thesynthetic PRP.

Another object of the invention is to provide compounds useful asintermediates in the preparation of synthetic PRP and methods ofpreparing such compounds.

Additional objects and advantages of the invention will be set forth inpart in the description that follows, and in part will be obvious fromthe description, or may be learned by the practice of the invention. Theobjects and advantages of the invention will be attained by means of theinstrumentalities and combinations particularly pointed out in theappended claims.

To achieve the objects and in accordance with the purpose of theinvention, as embodied and broadly described herein, the presentinvention provides an immunogenic oligosaccharide-protein conjugateuseful in a vaccine for protecting a mammal against H. influenzae. Theconjugate is made up of a PRP fragment, preferably a syntheticoligosaccharide, coupled to an H. influenzae adhesin protein.Preferably, the oligosaccharides contain from 2-30 ribosylribitolphosphate monomers, and from 1-30 of such oligosaccharides are attachedto the protein. In an alternative embodiment, the oligosaccharide isbound to a polypeptide that is an active site of the adhesin protein.

Preferably, the conjugate is represented by the following formula:##STR2## where m is 1-30, n is 2-30, R is (CH₂)_(p) CH₂ NH or (CH₂ CH₂O)_(p) CH₂ CH₂ NHCSNH where p is an integer from 1-3, and X is an H.influenzae adhesin protein or a fragment thereof containing an activesite of the protein.

The vaccine comprises an immunologically effective amount of theconjugate in a pharmaceutically acceptable carrier. Preferably, thevaccine also contains an adjuvant. The administration of the vaccine orconjugate to a human or other mammal induces a T-cell dependentprotective immune response.

The invention further comprises an isolated and a purified H. influenzaeadhesin protein and modified proteins and polypeptides derived from theadhesin protein, provided such derived proteins and polypeptides areimmunologically cross-reactive with the adhesin protein. Preferably,such derivatives are one or more epitopes of the adhesin protein. In aparticularly preferred embodiment, the epitope is also a receptorbinding site. The proteins and polypeptides may also be used in vaccineswithout being conjugated to the synthetic PRP.

In one embodiment, the adhesin protein is a minor H. influenzae outermembrane protein with a molecular weight of about 41,000 daltons. Inanother preferred embodiment, the adhesin protein is an H. influenzaeouter membrane protein with a molecular weight of about 47,000 daltons.

In one embodiment, the adhesin protein is purified from H. influenzaebacteria. Hi membranes are solubilized. The solubilized materialcontains the adhesin protein. This material is separated from theinsoluble material and contacted with receptors for the adhesin proteinfor period of time sufficient for the protein molecules to bind to thereceptors. The receptors are attached to an insoluble solid support. Asa result, the protein is separated from the solubilized material. Theprotein molecules are then removed from the receptors thereby beingrecovered in purified form.

In another embodiment, the adhesin proteins and related polypeptides ofthe invention are preferably recombinant proteins and polypeptides thathave been produced through genetic engineering techniques. They areproduced by an appropriate host cell that has been transformed by DNAthat codes for such proteins or polypeptides.

An isolated or substantially pure DNA sequence that codes for theadhesin proteins of the invention is obtained as follows. Adhesinprotein receptors or antibodies to the adhesin, preferably monoclonalantibodies, are used to screen a genomic library containing H.influenzae DNA. The library is made of clones which contain differentsequences of the DNA which have been operably and recoverably insertedinto a vector, with each of the vectors containing only one sequence ofthe DNA. The monoclonal antibodies or receptors identify the clones thatproduce the adhesin. The clone is then isolated. Preferably, theexogenous DNA sequences are recovered from the clone.

The invention further comprises isolated or substantially purified DNAderived from this DNA, for example, by single or multiple mutations.Preferably, such DNA hybridizes with the DNA obtained from the genomiclibrary under conditions of high stringency.

The invention further comprises a synthetic PRP oligosacchariderepresented by the following formula: ##STR3## where n is an integerfrom 2 to 30 and R¹ is (CH₂)_(p) CHO or (CH₂ CH₂ O)_(p) CH₂ CH₂ NH₂where p is an integer from 1 to 3.

In still another embodiment, the invention provides a compound useful asan intermediate in the preparation of synthetic PRP of the invention. Itis represented by the formula: ##STR4## where n is an integer from 2 to30, Bn is benzyl, and R² is (CH₂)_(p) CH(OR³)₂ or (CH₂ CH₂ O)_(p) CH₂CH₂ R⁴ where p is an integer from 1 to 3, R³ is an alkyl group 1-4carbons in length, and R⁴ is a group that can be converted into an aminogroup.

This compound is prepared using a solid phase synthesis. The monomer forchain initiation is a compound represented by the following formula:##STR5## where Bn is benzyl and MMTr is monomethoxytrityl. This monomeris coupled to a solid phase and then detritylated. The resultingdetritylated compound is coupled with a monomer for chain elongationrepresented by the formula: ##STR6## where Bn is benzyl and MMTr ismonomethoxytrityl. The resulting compound is then detritylated. Thechain elongation and detritylation steps are repeated a sufficientnumber of times until an oligomer of the desired length is obtained. Thechain terminating monomer is then added. The chain terminating monomeris represented by the formula: ##STR7## where Bn is benzyl and R² is(CH₂)_(p) CH(OR³)₂ or (CH₂ CH₂ O)_(p) CH₂ CH₂ R⁴ where p is an integerfrom 1 to 3, R³ is an alkyl group 1-4 carbons in length, and R⁴ is agroup that can be converted into an amino group. The phosphonate groupsof the support-bound oligomer are then oxidized to form phosphategroups. The resulting compound is then removed from the solid supportand recovered.

The protective groups on this intermediate are then removed byhydrogenation. Where R² is (CH₂ CH₂ O)_(p) CH₂ CH₂ R⁴, this results inthe synthetic PRP of the invention. In the case where R² is (CH₂)_(p)CH(OR³)₂, the hydrogenated compound is further subjected to selectiveacid hydrolysis.

The preferred conjugate of the invention is then prepared by couplingthe synthetic PRP with the Hi adhesin protein by reductive aminationwhere R¹ is (CH₂)_(p) CHO or, where R¹ is (CH₂ CH₂ O)_(p) CH₂ CH₂ NH₂,by preparing the corresponding isothiocynate and then coupling theisothiocynate with the protein.

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one embodiment of the inventionand, together with the description, serve to explain the principles ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an analysis of outer membrane preparations bySDS-polyacrylamide gel electrophoresis. Samples included the following(lanes): 1, total outer membrane protein preparation from Haemophilusinfluenzae type b stained with Coomassie blue; 2, autoradiography of ³⁵S-labeled total outer membrane proteins; 3, autoradiography of ³⁵S-labeled adhesin protein eluted from immobilized receptor asialo-GM₁ ;4, autoradiography of material eluted from immobilized globoside, anonsense glycolipid. Arrow indicates the adhesin migrating between P1and P2 with a molecular weight of about 41 kD.

FIG. 2 shows the neutralization of Haemophilus adhesin to the glycolipidreceptor asialo-GM₁. ³⁵ S!methionine-labeled membranes from Haemophilusinfluenza type b were incubated with serial dilutions of mouse sera andthen allowed to bind to receptor (0.5 microgram/well). The mouse seraused was obtained from 5 mice, designated M-0 through M5, which had beenimmunized with Haemophilus membranes. The sera from an unchallengedmouse (NMS) was used as a negative control.

FIG. 3 shows inhibition of Haemophilus membrane binding to asialo-GM₁with selected monoclonal antibodies. ³⁵ S!methionine-labeled membranesfrom Haemophilus were incubated with supernatants of hybridoma culturesand then allowed to bind to receptor (0.5 microgram/well). A negativereceptor control of Gb₄ indicates the specificity of the receptor-ligandinteraction. Mouse sera (M-2) (1:500 dilution) used in FIG. 2 showsstrong, positive inhibition. Media shows no inhibition of binding bymembranes to asialo-GM₁. Two classes of positively inhibiting hybridomaswere found. Hib 10 shows total inhibition of binding. Hib 30 and Hib 43show partial (about 35%) inhibition. Most hybridoma cultures, such asHib 2, showed no inhibition. All hybridoma cultures tested for bindingreacted positively with membranes in an ELISA. Error bars are includedto demonstrate the variability between duplicate wells.

FIG. 4 shows the identification and characterization of the 47 kDaHaemophilus adhesin. The monoclonal antibody which partially inhibitedmembrane binding, Hib 43, was reacted on Western blot to identify themolecular weight of the protein it recognizes. Whole cells were runafter no proteinase K treatment or either treatment with proteinase Kprior to lysis in sample buffer or treatment with proteinase K afterlysis in sample buffer (reading from left to right). Non-treatmentidentifies the 47 kDa protein; treatment of whole cells by proteinase Kprior to lysis indicates the sensitivity of the protein to this proteasein its native location; and treatment after lysis by proteinase Kdemonstrates the general sensitivity to this protease after disruptionfrom that native location. The Escherichia coli XL-1, transformed withpMC101, expresses the 47 kDa Haemophilus protein, which reacts with Hib43. The 47 kDa protein was also sensitive to proteinase K treatment ofXL-1/pMC101 whole cells. These data suggest a surface location for thisprotein in both hosts.

FIG. 5 shows a restriction map of the region in Haemophilus influenzatype b that encodes the 47 kDa adhesin. A 10.5 kbp Eco R1 fragment thatproduces the 47 kDa protein which reacts with Hib 43 monoclonal antibodywas cloned from an Haemophilus lambda ZAPII genebank. The helper phageR408 was used to induce a plasmid containing this insert in the vectorpSK(-).

FIG. 6 shows the glycolipid binding phenotype of Escherichia coli thatexpress the Hib 47 kDa protein. The ability of membranes from the E.coli, XL-1, or from XL-1 transformed with pMC101, designated 3, werecompared using the standard binding assay. Serial dilutions were made ofglycolipids with receptor activity: asialo-GM₁, asialo-GM₂, sulfatide,or the negative control, Gb₄. XL-1/pMC101 binds with high affinity tothese receptors, similar to Haemophilus.

FIGS. 7A-7D show the nucleotide sequence of hin47 (SEQ ID NO:1) and thededuced Hin47 amino acid sequence (SEQ ID NO:2). The nucleotide sequenceis numbered above each line and the deduced Hin47 amino acid sequence isshown below the line. The open reading frame for the hin47 gene isbetween nucleotide 115 and 1503. The end of the putative leader sequenceand beginning of the putative mature polypeptide is indicated atnucleotide base 189. The predicted molecular weight of the maturepolypeptide is 46399 and it has a pI of 5.86.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention, which, together with the followingexamples, serve to explain the principles of the invention.

The invention comprises an immunogenic oligosaccharide-H. influenzaeadhesin protein conjugate useful as a vaccine against H. influenzae,purified H. influenzae adhesin proteins and related proteins andpolypeptides, DNA coding for the proteins and polypeptides, host cellscontaining the DNA and producing the proteins and polypeptides,synthetic PRP oligosaccharides and intermediates useful for theirsynthesis, and methods of making and using these materials.

Immunogenic Conjugate

The conjugate comprises a polyribosylribitol phosphate fragmentchemically coupled to a purified H. influenzae adhesin protein.Preferably, the PRP fragment is a synthetic oligosaccharide. From 1 toabout 30 and preferably from about 5 to 20 of the natural fragments orsynthetic oligosaccharides are attached to the protein.

The fragments are attached to the protein by known techniques forcovalently attaching polysaccharides to proteins or polypeptides,applied to the teachings contained herein. The preferred techniques hereare reductive amination or isothiocyanate coupling.

Any adhesin protein may be used. In one embodiment, the purified adhesinprotein is a minor Hi outer membrane protein with a molecular weight ofabout 41,000 daltons, distinct from P1 or P2.

In another preferred embodiment, the purified adhesin protein is an Hiouter membrane protein with a molecular weight of about 47,000 daltons,distinct from P1-P6.

Alternatively, the protein may be replaced by a polypeptide that is anactive site of the adhesin protein. As used herein, the term "activesite" means an epitope (antigenic determinant) or an H. influenzaereceptor binding site, which may or may not also be an epitope. As usedherein, the term "receptor" is a macromolecule that binds to an Hiadhesin protein. The macromolecule is preferably a glycosphingolipid.Without intending to limit the scope of the invention, it is believedthat the binding site is an epitope.

When the PRP fragment is obtained from natural sources, it is of varyinglengths, but preferably about 8 to 120 monomers in length. Suchfragments are obtained by known techniques, such as those described inthe above-referenced European Patent Office Publication No. 0 338 265.

Synthetic PRP is a linear homopolymer of alternating molecules of riboseand ribitol joined by a phosphodiester linkage and represented by theformula: ##STR8## where n is 2 to 30 and preferably 5-20. Such syntheticPRP's include those known in the art as well as the novel ones of theinvention. For example, the previously mentioned European Patent OfficePublications 0 320 942 and 0 276 516 disclose synthetic PRP's that couldbe used in the conjugate of the invention.

Preferably, the synthetic PRP is a compound represented by the formula:##STR9## where n is an integer from 2 to 30 and R¹ is (CH₂)_(p) CHO or(CH₂ CH₂ O)_(p) CH₂ CH₂ NH₂ where p is an integer from 1 to 3.Preferably n is 5-20 and p is 1. The synthetic PRP will be associatedwith a counter ion. Preferably, the ion is sodium (Na⁺).

The synthetic oligosaccharides usually contain a chemical spacer orlinker by which they are attached to the protein. Such a spacer may beany chemical linkage that serves to connect the PRP and the protein andthat has limited or no adverse effect to the animal host when theconjugate is administered. Such spacers may include those known in theart as well as the novel spacers of the invention. Known spacers includethose disclosed in the previously mentioned European Patent OfficePublications 0 320 942 and 0 276 516 as well as those disclosed in U.S.Pat. No. 4,830,852 issued May 16, 1989 to Marburg, et al., the latter ofwhich is incorporated herein by reference. Preferably, the chemicalspacer is a moiety represented by the formula: ##STR10## where R is(CH₂)_(p) CH₂ NH or (CH₂ CH₂ O)_(p) CH₂ CH₂ NHCSNH and p is an integerfrom 1-3, preferably 1.

In the most preferred embodiment of the invention, the conjugate isrepresented by the following formula: ##STR11## where m is 1-30, n is2-30, R is (CH₂)_(p) CH₂ NH or (CH₂ CH₂ O)_(p) CH₂ CH₂ NHCSNH where p isinteger from 1-3, and X is an H. influenzae adhesin protein or afragment thereof containing an active site of the protein. Preferably, mis 5-20, n is 5-20, and p is 1. The symbol X in the above-referencedformula may also represent certain derived or modified proteins orpolypeptides discussed below. The conjugate will be associated with acounter ion. Preferably, the ion is Na⁺.

Adhesin Proteins

The invention further comprises an isolated H. influenzae adhesinprotein. As used herein, in this context, the term "isolated" means thatthe protein is significantly free of other proteins. That is, acomposition comprising the isolated protein is between 70% and 94% pureby weight. Preferably, the protein is purified. As used herein, the term"purified" and related terms means that the protein is at least 95% pureby weight, preferably at least 98% pure by weight, and most preferablyat least 99% pure by weight. The protein binds to a receptor selectedfrom the group consisting of fucosylasialo-GM₁, asialo-GM₁, andasialo-GM₂, all of which contain the structureN-acetylgalactosamine(beta 1-4)galactose(beta 1-4)glucose-(beta1-1)ceramide abbreviated GalNAc(betal-4)Gal(betal-4)Glc(betal-1)Cer. Theprotein also binds to another receptor, phosphatidylethanolamine.

In one embodiment, the protein is a minor outer membrane protein with amolecular weight of about 41 KD as determined by SDS PAGE. It isdistinguishable from the various major outer membrane proteins that havebeen identified for Hi. In particular, the protein appears as a fainterband between the bands on a polyacrylamide gel for the outer membraneproteins known as P1 and P2. See FIG. 1.

This purified Hi adhesin protein is prepared preferably from naturalsources as follows. Hi bacterial membranes are obtained by standardtechniques and solubilized, using a solubilizing compound, such as adetergent. Preferably, the membranes are mixed with the detergent, andthe mixture is sonicated. The most preferred solubilizing agent is asolution containing about 1.0% to about 1.5% and preferably about 1.3%octylglucopyranoside. The adhesin protein is in the solubilizedmaterial. The remaining insoluble material from the membrane isseparated, preferably by centrifuging.

The supernatant is contacted with receptors that bind the protein andare attached to an insoluble solid support or matrix, such as amicrotiter well or a gel, for a period of time and under conditionssufficient for the protein to bind to the receptors, thus separating theprotein from the other material. The preferred receptors for the adhesinprotein are fucosylasialo-GM₁, asialo-GM₁, asialo-GM₂, andphosphatidylethanolamine. These receptors can be prepared in accordancewith the procedures disclosed in Krivan, et al., Proc. Natl. Acad. Sci.USA, 85:6157-6161 (1988), incorporated herein by reference. The mostpreferred receptor, asialo-GM₁, is also commercially available. All ofthese receptors, except phosphatidylethanolamine, contain thecarbohydrate sequence GalNAc(betal-4)Gal(betal-4)Glc, which,accordingly, may also be used as a receptor for the purification of theadhesin protein. This sequence can be prepared using standardcarbohydrate synthesis techniques.

The adhesin protein is then eluted using the appropriate agent. This maybe free receptor in solution, SDS elution buffer, or a chaotropic agent,such as KSCN, NaCl, or quanidine hydrochloride. The eluted protein isthen tested against the receptor to confirm that the protein does bindto it. The purity of the isolated protein is analyzed by SDS-PAGE.Preferably, it will be about 99% pure after affinity purification withthe most preferred receptor.

For purification of larger amounts of the adhesin protein,chromatography is preferred. The receptor is immobilized onto ahydrophobic gel support, such as octylagarose. This matrix is preparedby adsorbing the receptors to the hydrophobic gel in the presence ofsalt as described by Hirabayashi, et al. for other glycolipids.Hirabayashi, et al., J. Biochem., 94:327-330 (1983), incorporated hereinby reference. Photoactivatable heterobifunctional crosslinking agentshave also been used to prepare glycolipid affinity matrices. Lingwood,C., J. Lipid Res., 25:1010-1012 (1984), incorporated herein byreference. In this case, the receptor-active lipid is covalentlycrosslinked to the gel support. The column is then preferably washedextensively with an appropriate buffer solution, such as TMS-buffersaline, before the protein is eluted.

A more preferred method is to purify the adhesin by affinitychromatography using an anti-adhesin monoclonal or polyclonal antibodyprepared by standard techniques. In this case, the antibodies arecovalently linked to agarose gels activated by cyanogen bromide orsuccinamide esters (Affi-Gel, BioRad Inc.) or by other methods known bythose skilled in the art. The sonic extract is loaded on the top of thegel as described above.

In another preferred embodiment, the adhesin proteins comprise an H.influenzae outer membrane protein with a molecular weight of about47,000 daltons. FIGS. 7A and 7B show the protein amino acid sequence aswell as the designated nucleotide sequence of the open reading frame(ORF) encoding a 49 kDa protein. The 49 kDa protein comprises 463 aminoacids (amino acids 1-463 in FIG. 7A and 7B), includes a putative signalsequence of approximately 2.5 kDa and 25 amino acids, thereby resultingin a mature protein of approximately 47 kDa and 438 amino acids (aminoacids 26 through 463 on FIG. 7A and B), herein designated Hin47. Thisprotein is distinguishable from the known Hi proteins P1-P6 on the basisof molecular weight and the fact that those proteins are integralmembrane proteins, while this protein is an outer membrane protein. Thisprotein also binds to the previous mentioned receptors as well as tosulfatide, (SO₃ ⁻ -galactose(beta 1-1)ceramide) and it is soluble in 1%Sarkosyl (N-lauroylsarcosine).

This protein is preferably prepared in purified form as follows. Himembranes are extracted with a solution that removes membrane associatedproteins, which produces an extract containing the adhesin protein alongwith other membrane associated proteins. Preferably, this solution is anonionic detergent, such as Sarkosyl or octylglucopyranoside. Theinsoluble material is separated from the extract, preferably bycentrifugation. This produces a supernatant that contains the adhesinprotein. The supernatant is then brought into contact with a monoclonalantibody which recognizes the adhesin protein. The antibody is bound toan insoluble solid support. The contact is for a period of time andunder standard reaction conditions sufficient for the adhesin protein tobind to the monoclonal antibody. Preferably, the solid support is amaterial used in a chromatographic column. The adhesin protein is thenremoved from the antibody, thereby permitting the recovery of protein inpurified form. Preferably, the nonionic detergent solution is removedfrom the supernatant before the supernatant is subjected to the affinitychromatography. Such removal is preferably accomplished by dialyzing thesupernatant to produce a dialysate that is substantially free of thedetergent.

The monoclonal antibodies can be prepared by standard techniques, giventhe teachings contained herein. Such techniques are disclosed, forexample, in U.S. Pat. No. 4,271,145, issued Jun. 2, 1981 to Wands et al.and U.S. Pat. No. 4,196,265, issued Apr. 1, 1980 to Koprowski et al.,both of which are herein by reference. Briefly, mice are immunized withHi membranes. Hybridomas are prepared by fusing spleen cells from themice with myeloma cells. The fusion products are screened for thoseproducing antibodies that bind to the Hi membranes. The positive clonesare then screened to identify those whose binding with the Hi membranesis inhibited by an Hi adhesin receptor. The positive hybridomas clonesare isolated, and the monoclonal antibodies are recovered from thoseclones.

Alternatively, the outer membrane proteins could be separated on a gel.The 47 kDa band could be cut out and injected into the mice. Thehybridomas could be prepared and screened as described above.

DNA

The adhesin proteins of the invention are preferably produced throughgenetic engineering techniques. In this case, they are produced by anappropriate host cell that has been transformed by DNA that codes forthe proteins. Preferably, the host cell is a bacterium, and mostpreferably the bacterium is E. coli, B. subtilis, or Salmonella.

The DNA of the invention is an isolated or substantially purified DNAsequence (i.e., polydeoxyribonucleotide molecule) encoding a protein orpolypeptide that binds to the previously mentioned receptors.Preferably, the DNA of the invention includes an open reading frame(ORF) sequence (nucleotides 115 through 1503 in FIGS. 7A and B),designated hin47, encoding an approximate 49 kDa and 463 amino acidprotein, designated Hin47, as shown in FIGS. 7A and B. Most preferably,the DNA comprises that part of the ORF that does not code for the signalsequence (nucleotides 191 through 1503 in FIGS. 7A and B). As usedherein, the term "isolated" and variations thereof means that the DNA isin isolation from DNA encoding other proteins or polypeptides normallyaccompanying the Hi adhesin proteins. Thus, the DNA of the inventionincludes DNA encoding the protein or polypeptide when that DNA has beencloned into a microbial vector, such as a plasmid, or into a viralvector that may be harbored by a bacteriophage, provided that suchclones are isolated from clones that contain DNA encoding other proteinsor polypeptides normally accompanying this one. As used herein, the term"substantially pure" and variants thereof means that the DNA issubstantially free of DNA and RNA that does not encode the proteins orpolypeptides of the invention. That is, there will be no more than about1% by weight of other DNA and RNA and preferably no more than about 0.2%by weight of other DNA and RNA in any sample that contains the DNA ofthe invention.

Preferably, the DNA is obtained by using either the receptors ormonoclonal antibodies to the adhesins to screen an appropriate genomiclibrary that contains H. influenzae DNA. Such a library comprisescolonies of a single type of microorganism, generally bacteria like E.coli K12 (XL-1), into which pieces of the foreign DNA have beeninserted, generally by being incorporated into a plasmid, cosmid, orphage vector compatible with the microorganism. More specifically, thelibrary comprises clones of vectors into which different sequences ofthe DNA have been operably and recoverably inserted, each of the vectorscontaining only one sequence of the DNA. The vectors may be plasmids,cosmids, phagemids, or phage genomes. If necessary because of the typeof library being used, segments of DNA will have been inserted into thevectors in a manner that they will be expressed under appropriateconditions (i.e., in proper orientation and correct reading frame andwith appropriate expression sequences, including an RNA polymerasebinding sequence and a ribosomal binding sequence.) The microorganismswill be ones that do not express the adhesin protein, such as E. coliHB101.

Clones from the library are brought into contact with the receptors orantibodies to identify those clones that bind. The clones are isolatedand the exogenous DNA sequence is recovered from one of the clones. Thesequence is preferably evaluated to determine if it encodes the protein.

Preferably, the genomic library comprises bacteria, such as E. coliinfected by phage, preferably bacteriophage lambda. Plaques produced bythe phage infected bacteria are screened by monoclonal antibodies toidentify those plaques containing bacteria that produce the adhesinprotein. The screening involves contacting the plaques with themonoclonal antibody to determine if binding has occurred, using standardtechniques. Preferably, immunoassays are used.

In this preferred embodiment, the positive clones are then isolated bypurifying the positive plaques and inducing plasmid formation in thebacteria in the purified plaque with a helper phage according tostandard techniques.

In an alternate preferred embodiment, colonies containing DNA thatencodes an Hi adhesin protein could be detected using DYNA Beadsaccording to Olsvick et al., 29th ICAAC, Houston, Tex. 1989,incorporated herein by reference. The previously described receptorswould be crosslinked to tosylated DYNA Beads M280, and thesereceptor-containing beads would then be used to adsorb to coloniesexpressing the adhesin protein. Colonies not expressing the adhesinwould be removed by washing, and this process would be repeated toobtain an appropriate enrichment. Putative adhesin expressing colonieswould then be plated and confirmed by metabolically labeling each colonywith ³⁵ S-methionine and testing the ability of the colony to bind tothe receptor as previously described. The DNA from several adheringclones would be compared to identify shared sequences, and these sharedsequences would be further subcloned and characterized.

Alternatively, the receptors could be nonspecifically immobilized to asuitable support, such as silica or Sealite resin. This material wouldthen be used to adsorb to colonies expressing the adhesin protein asdescribed in the preceding paragraph.

In another alternate preferred embodiment, the gene for a specificadhesin would be localized and identified by constructing non-adherentmutants of a specific pathogen. This would be accomplished by creatingmutants using a transposable element such as TnPhoA as described inManoil et al., Proc. Natl. Acad. Sci. USA, 82:81129-81133 (1985),incorporated herein by reference. Alkaline phosphatase positive mutantswould indicate mutations within exported proteins. Since the adhesin foreach pathogen is located on the outer membrane surface and thereforeexported, this set of mutants would contain a much reduced subset ofmutants. They would then be screened for a loss in binding activity.

It will be recognized by persons skilled in the art that a DNA sequencefor an Hi adhesin protein can be modified by known techniques in view ofthe teachings disclosed herein. For example, different codons can besubstituted that code for the same amino acid as the original codon.Alternatively, the substitute codons may code for a different amino acidthat will not affect the immunogenicity of the protein or which mayimprove its immunogenicity. For example, oligonucleotide directed, sitespecific mutagenesis or other techniques to create single or multiplemutations, such as replacements, insertions, deletions, andtranspositions, as described in Botstein and Shortle, "Strategies andApplications of In vitro Mutagenesis," Science, 229:1193-1210 (1985),which is incorporated herein by reference, can be employed. Since suchmodified DNA can be obtained by the application of known techniques tothe teachings contained herein, such DNA is within the scope of theclaimed invention.

Moreover, it will be recognized by those skilled in the art that the DNAsequence (or fragments thereof) of the invention can be used to obtainother DNA sequences that hybridize with it under conditions of moderateto high stringency (including the derived sequences discussed in thepreceding paragraph), using general techniques known in the art. Thatis, the hybridizing sequences are at least 90% homologous and preferablyat least 95% homologous to hin47. Accordingly, the DNA of the inventionincludes such DNA.

The DNA of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform a microorganism for the expression and production of theadhesins of the invention. Such techniques include those disclosed inU.S. Pat. Nos. 4,440,859 issued Apr. 3, 1984 to Rutter et al., U.S. Pat.No. 4,530,901 issued Jul. 23, 1985 to Weissman, U.S. Pat. No. 4,582,800issued Apr. 15, 1986 to Crowl, U.S. Pat. No. 4,677,063 issued Jun. 30,1987 to Mark et al., U.S. Pat. No. 4,678,751 issued Jul. 7, 1987 toGoeddel, U.S. Pat. No. 4,704,362 issued Nov. 3, 1987 to Itakura et al.,U.S. Pat. No. 4,710,463 issued Dec. 1, 1987 to Murray, U.S. Pat. No.4,757,006 issued Jul. 12, 1988 to Toole, Jr., et al., U.S. Pat. No.4,766,075 issued Aug. 23, 1988 to Goeddel, et al., and U.S. Pat. No.4,810,648 issued Mar. 7, 1989 to Stalker, all of which are incorporatedherein by reference.

The DNA of the invention may be joined to a wide variety of other DNAsequences for introduction into an appropriate host cell. The companionDNA would depend upon the nature of the host cell, the manner of theintroduction of the DNA into the host cell, and whether episomalmaintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques.

Generally, not all of the hosts will be transformed by the vector.Therefore, it will be necessary to select for transformed host cells.Once selection technique involves incorporating into the expressionvector a DNA sequence, with any necessary control elements, that codesfor a selectable trait in the transformed cell, such as antibioticresistance. Alternatively, the gene for such selectable trait can be onanother vector, which is used to co-transform the desired host cell. Thepreferred expression vector for use in the invention is the plasmidpMC101. The preferred host cell is E. coli.

The transformed host cells express the proteins or polypeptides of theinvention. Such cells are cultured by known techniques, and the proteinsor polypeptides are recovered by known techniques. Depending upon thehost and expression system used, the recombinant proteins andpolypeptides of the invention may be part of a fusion protein producedby the transformed host cells. Such proteins are recovered by knowntechniques, and the undesired part may be removed by known techniques.Alternatively, the fusion protein itself may be more immunogenic thanthe recombinant protein or polypeptide alone and, therefore, may itselfbe used in a vaccine.

If desirable, the adhesins can be further purified by the application ofstandard protein purification techniques, modified and applied inaccordance with the discoveries and teachings described herein. Suchtechniques include electrophoresis, centrifugation, gel filtration,precipitation, dialysis, chromatography (including ion exchangechromatography, affinity chromatography, immunoadsorbent affinitychromatography, reverse-phase high performance liquid chromatography,and gel permeation high performance liquid chromatography), isoelectricfocusing, and variations and combinations thereof.

One or more of these techniques are employed sequentially in a proceduredesigned to separate molecules according to their physical or chemicalcharacteristics. These characteristics include the hydrophobicity,charge, binding capability, and molecular weight of the protein. Thevarious fractions of materials obtained after each technique are testedfor their ability to react with the adhesin receptors. Those fractionsshowing such activity are then subjected to the next technique in thesequential procedure, and the new fractions are tested again. Theprocess is repeated until only one fraction reactive with the receptorsremains and that fraction produces only a single band when subjected topolyacrylamide gel electrophoresis.

The preferred techniques include those identified and described in U.S.Pat. No. 4,446,122 issued May 1, 1984 to Chu, et al., which isincorporated herein by reference. Preferably, the adhesins are purifiedby receptor affinity chromatography or antibody affinity chromatography.

Modified Adhesins

The adhesins of the invention may be modified by known proteinmodification techniques. Such modifications include breaking the proteininto fragments that contain at least one active site or the addition,substitution, or deletion of one or more amino acids to the protein or afragment thereof. Preferably, such derived proteins or polypeptides areimmunologically cross-reactive with the Hi adhesin proteins, thus beingcapable of eliciting an antigenic response to H. influenzae in an animalhost. Most preferably, such derived proteins or polypeptides also bindto an H. influenzae receptor selected from the group consisting offucosylasialo-GM₁, asialo-GM₁, and asialo-GM₂. (As used in thisspecification, the term "polypeptide" also includes shorter chains ofamino acids that are often referred to as peptides.) Such modificationsmay enhance the immunogenicity of the protein or have no effect on suchactivity. The modification techniques include those disclosed in U.S.Pat. No. 4,526,716, issued Jul. 2, 1985 to Stevens, incorporated hereinby reference.

The proteins of the invention may contain one or more amino acidsequences that are not necessary to their immunogenicity. It may be thecase, for example, that only the amino acid sequences of a particularepitope of the antigen will be necessary for immunogenic activity.Unwanted sequences can be removed by techniques well-known in the art.For example, unwanted amino acid sequences can be removed via limitedproteolytic digestion using enzymes such as trypsin, papain, or relatedproteolytic enzymes.

This latter approach is expected to be particularly useful for theadhesin protein of the invention. Since the protein binds to severalrelated receptors having a consensus sequence, the protein should have awell conserved region that acts as the receptor binding site. This siteis the particularly preferred polypeptide of the invention.

Alternatively, polypeptides corresponding to various immunogenicepitopes and/or the receptor binding site of the protein may bechemically synthesized by methods well-known in the art, given theteachings contained herein. These include the methods disclosed in U.S.Pat. No. 4,290,944, issued Sept. 22, 1981 to Goldberg, incorporatedherein by reference.

Modified proteins or polypeptides can be prepared that are substantiallyhomologous to the Hi adhesin protein or to the polypeptides discussedabove through the use of known techniques and routine experimentation inview of the teachings contained herein. As used herein, the term"substantially homologous" means immunologically cross-reactive. Such aprotein or polypeptide may be identified by the fact that it will bindto antibodies that were made to the adhesin protein of the invention,which antibodies can be prepared by standard techniques. Some of suchmodified proteins or polypeptides may have enhanced immunogenicitycompared to the one from which they are derived.

Thus, the invention includes a class of derived proteins andpolypeptides, including synthetically derived peptides or fragments ofthe adhesin protein, having common elements of origin, structure, andmechanism of action, such as immunogenic effect or being able to bind tothe previously mentioned receptors, that are within the scope of thepresent invention because they can be prepared by persons skilled in theart without undue experimentation, once given the teachings of thepresent invention. Moreover, since persons skilled in the art can makemodifications to or derivatives of epitopes or the receptor binding siteon the proteins or polypeptides of the invention, once such epitopes orsite are identified, such modifications or derivatives are within thescope of the invention. Such derived proteins and polypeptides arepreferably pure as that term was previously defined herein.

The Hi adhesin protein of the invention (as well as the related proteinsand polypeptides derived therefrom) has utility not only in theconjugate vaccine but as an immunogen in its own right. Thus, it can beused in a vaccine for animals, including mammals, rodents, primates, andhumans. The preferred use is a vaccine for humans, preferably children,and most preferably young infants.

Such a vaccine can be prepared by techniques known to those skilled inthe art and would comprise, for example, the antigen, a pharmaceuticallyacceptable carrier, an appropriate adjuvant, and other materialstraditionally found in vaccines. An immunologically effective amount ofthe antigen to be used in the vaccine is determined by means known inthe art in view of the teachings herein.

Synthetic PRP

The invention further comprises novel synthetic PRP represented by theformula: ##STR12## where n is an integer from 2 to 30, preferably 5-20,and R¹ is (CH₂)_(p) CHO or (CH₂ CH₂ O)_(p) CH₂ CH₂ NH₂ where p is aninteger from 1 to 3, preferably 1. The ability to prepare this novelsynthetic PRP permits the preparation of compositions where all of thePRP oligosaccharides are of the same length (i.e., have the same numberof monomeric units), in contrast to PRP obtained from natural sources,where the fragments vary tremendously in length.

The PRP of the invention is prepared by a combination of solid phasesynthesis and the highly efficient H-phosphonate method for theconstruction of the phosphodiester linkage. It also involves the use ofgels with higher levels of functionalization, which are better suitedfor commercial scale operations.

The general approach is to prepare a protected oligomeric ribosylribitolphosphate derivative by the following steps. First, the monomer forchain initiation is coupled to a solid phase. The monomer is representedby the formula: ##STR13## where Bn is benzyl and MMTr ismonomethoxytrityl. See Compound 7, Table 1. The preferred solid phase isa Merrifield-type amino resin. The chain initiation monomer (Compound 7)is coupled to the solid phase by known techniques, such as reaction withsuccinic anhydride, followed by coupling of the obtained succinate ofCompound 7 to amino groups of the solid phase. The loading is determinedby colorimetric quantification of the trityl cation released on acidtreatment. The coupled compound is then detritylated, such as bytreatment with trifluoroacetic acid in dichloromethane.

Chain elongation is accomplished by coupling the detritylated chaininitiation monomer with a compound represented by the formula: ##STR14##where Bn is benzyl and MMTr is monomethoxytrityl. See Compound 8,Table 1. (The compound will be associated with a counter ion.Preferably, the ion is an organic cation, such as triethyl ammonium.)The coupling is accomplished by using a condensing reagent, such aspivaloyl chloride. The resulting compound is then detritylated. Thechain elongation-detritylation steps are repeated a sufficient number oftimes to prepare an oligosaccharide of the desired length. Thus, if nrepresents the desired number of PRP monomers in the oligosaccharide,the chain elongation-detritylation cycles are repeated n-2 times afterthe coupling of the chain initiation monomer and the first chainelongation monomer.

The chain is terminated by coupling it with a chain termination monomerrepresented by the following formula: ##STR15## where Bn is benzyl andR² is (CH₂)_(p) CH(OR³)₂ or (CH₂ CH₂ O)_(p) CH₂ CH₂ R⁴ where p is 1-3,R³ is an alkyl group 1-4 carbons in length, and R⁴ is a group that canbe converted into an amino group. See Compounds 10 and 12, Table 2. (Thecompound will be associated with a counter ion. Preferably, the ion isan organic cation, such as triethyl ammonium.) Preferably, p is 1, andR³ is methyl or ethyl. Preferably, R⁴ is N₃, trifluoroacetyl,benzyloxycarbonyl, or fluorenylmethoxycarbonyl.

The phosphonate groups of the solid-bound oligomer are then oxidized toform phosphate groups. Preferably, this is accomplished by treatmentwith iodine in aqueous pyridine.

The resulting compound is then removed from this solid support,preferably through cleavage by methanolysis. The recovered compound isrepresented by the formula: ##STR16## where n is an integer from 2 to30, preferably 5-20, Bn is benzyl, and R² is defined as above. SeeCompounds 13 and 15, Table 3. (The compound will be associated with acounter ion. Preferably, the ion is ammonium or substituted ammonium.)

The resulting compound is then deprotected by hydrogenation withpalladium on charcoal. In the case where R² is (CH₂)_(p) CH(OR³)₂, thehydrogenated compound is further subjected to selective acid hydrolysis,such as by treatment with aqueous trifluoroacetic acid. The resultingPRP oligomers are purified by standard techniques, preferably byion-exchange chromatography, HPLC or gel filtration. See Compounds 14and 16, Table 3.

Table 1 shows the synthesis of the chain initiation monomer, Compound 7,and the chain elongation monomer, Compound 8. The readily availablemethyl 2,3-isopropylidene-beta-D-ribofuranoside (Compound 1) (Leonard,et al., J. Het. Chem. 3:485 (1966), incorporated herein by reference) isused as starting material. Allylation of Compound 1 with allylbromide/sodium hydroxide in N,N-dimethylformamide gives the expected5-O-allyl Compound 2 as an oil that can be distilled. This compound issubjected to a sequence of reactions comprising hydrolysis with aqueousformic acid, sodium borohydride reduction, tritylation withtriphenylmethylchloride/pyridine, benzylation with benzylchloride/sodium hydroxide in N,N-dimethylformamide, and hydrolysis withaqueous acetic acid. The resulting Compound 3 is purified by silica gelchromatography.

Benzylation of Compound 1 with benzyl chloride/sodium hydroxide inN,N-dimethylformamide gives the expected 5-O-benzyl compound 4 as an oilthat can be distilled. This compound is subjected to a sequence ofreactions comprising hydrolysis with aqueous formic acid andbenzoylation with benzoyl chloride in pyridine, giving Compound 5, whichis purified by chromatography and crystallization. Compound 5 issubjected to a further sequence of reactions comprising treatment withhydrogen bromide in dichloromethane to prepare the glycosyl bromide,followed by treatment with methanol and collidine. The resultingorthoester is then debenzoylated with sodium methoxide in methanol. Theresulting product is allylated with allyl bromide/sodium hydroxide inN,N-dimethylformamide to give, after purification by silica gelchromatography, Compound 6.

Glycosylation can be accomplished by several methods. In the preferredmethod (A), Compound 6 is treated with trimethylsilyl chloride to givethe corresponding glycosyl chloride, which, when treated with Compound 3in the presence of molecular sieves, gives a ribitol glycoside.Alternatively (B), Compound 6 is transesterified in the presence ofCompound 3. The resulting ribitol orthoester is then rearranged in situto give the ribitol glycoside.

The ribitol glycoside is then subjected to debenzoylation with sodiummethoxide in methanol and benzylation with benzyl chloride/sodiumhydroxide in N,N-dimethylformamide. The resulting5-O-allyl-2,3,4-tri-O-benzyl-1-O-(3-O-allyl-2,5-di-O-benzyl-beta-D-ribofuranosyl)-D-ribitolis deallylated by treatment with, successively,tris-(triphenylphosphine)rhodium(I)chloride and aqueous acetic acid andmonomethoxytritylated with monomethoxytrityl chloride. The resultingchain initiation monomer (Compound 7) is purified by chromatography.

The condensation reaction of Compound 7 with phosphorousacid/5,5-dimethyl-2-oxo-2-chloro-1,3,2-dioxaphosphorinane gives thechain elongation monomer (Compound 8).

Table 2 shows the synthesis of the monomers for chain termination.Compound 6 is reacted with trimethylsilyl chloride to give thecorresponding chloride, which is reacted with the appropriate alcoholsin the presence of molecular sieves to give beta-glycosides of thealcohols. Preferably, the alcohols are 2-(2-azidoethoxy)ethanol, 2-2-benzyloxycarbonylamido)ethoxy!ethanol, or 2,2-diethoxyethanol. Thebeta-glycosides are subjected to the reaction sequence debenzoylation,benzylation, and deallylation, as in the preparation of Compound 7,which gives Compounds 9 or 11. Condensation with phosphorousacid/5-5-dimethyl-2-oxo-2-chloro-1,2,3-dioxa-phosphorinane according tothe same procedure used to prepare Compound 8 gives the desiredspacer-containing monomers (Compounds 10 or 12).

Table 3 shows the specific PRP oligomers obtained after solid phasesynthesis employing Compounds 7, 8, and 10 or 12. Compounds 13 and 15are the protected oligomers after removal from the solid support, andCompounds 14 and 16 are the final oligomers after deprotection.

The preferred use of the novel PRP is in the preparation of the novelimmunogenic conjugates. The oligomer is coupled to one of the proteinsor polypeptides of the invention by standard techniques applied to theteachings contained herein. When the spacer terminates in an aldehydegroup, the preferred technique is reductive amination using sodiumborohydride as described in Roy, et al., J. Carbohydr. Chem. 6:161-165(1987) and Lee, et al., Carbohydr. Res., 77:149-156 (1979), both ofwhich are incorporated by reference. When the spacer terminates with anamino group, the PRP is converted into the isothiocynate by treatmentwith an activated thiocarbonic acid derivative, such as thiophosgene,and then coupled to the protein at a pH of 9-10 in accordance with theprocedures described in Kallin, et al., Glycoconjugate J., 3:311-319(1986) and Zopf, et al., Methods Enzymol., 50:171-175 (1978), both ofwhich are incorporated herein by reference. The ratio ofprotein/carbohydrate is determined by a combination of Lowry proteindetermination and ribose determination. The ratio is primarily afunction of the ratio of carbohydrate to protein in the initial reactionmixture and the type of spacer used. As shown in Example 3, the use of aspacer terminating in an amino group (Compound 16) results in a greaternumber of oligosaccharides being coupled to the protein than the use ofa spacer terminating in an aldehyde group (Compound 14). Table 4 showsthe formulas of the final conjugates.

Vaccines

The adhesin-oligosaccharide conjugates, as well as their proteincomponents as previously mentioned, may be used in vaccines against bothinvasive and non-invasive strains of H. influenzae. The conjugatevaccines should have greatest utility against H. influenzae type b.

The vaccines comprise an immunologically effective amount of theimmunogen in a pharmaceutically acceptable carrier. The combinedimmunogen and carrier may be an aqueous solution, emulsion, orsuspension. An immunologically effective amount is determinable by meansknown in the art without undue experimentation, given the teachingscontained herein. In general, the quantity of immunogen will be between0.1 and 100 micrograms per dose. The carriers are known to those skilledin the art and include stabilizers, diluents, and buffers. Suitablestabilizers include carbohydrates, such as sorbitol, lactose, manitol,starch, sucrose, dextran, and glucose and proteins, such as albumin orcasein. Suitable diluents include saline, Hanks Balanced Salts, andRingers solution. Suitable buffers include an alkali metal phosphate, analkali metal carbonate, or an alkaline earth metal carbonate. Thevaccine may also contain one or more adjuvants to improveimmunogenicity. Suitable adjuvents include aluminum hydroxide, aluminumphosphate, or aluminum oxide or a composition that consists of a mineraloil, such as Marcol 52, or a vegetable oil and one or more emulsifyingagents.

The vaccine may also contain other immunogens. Such a cocktail vaccinehas the advantage that immunity against several pathogens can beobtained by a single administration. Examples of other immunogens arethose used in the known DPT vaccines.

The vaccines of the invention are prepared by techniques known to thoseskilled in the art, given the teachings contained herein. Generally, theimmunogens are mixed with the carrier to form a solution, suspension, oremulsion. One or more of the additives discussed above may be in thecarrier or may be added subsequently. The vaccine preparations may bedessicated, for example, by freeze drying for storage purposes. If so,they may be subsequently reconstituted into liquid vaccines by theaddition of an appropriate liquid carrier.

The vaccines are administered to humans or other mammals, includingrodents and primates. Preferably, they are administered to humanchildren, most preferably children younger than 18 months of age. Theycan be administered in one or more doses. The vaccines may beadministered by known routes of administration for this type of vaccine.The preferred routes are intramuscular or subcutaneous injection.Accordingly, the invention also comprises a method for inducing animmune response to Hi in a mammal in order to protect the mammal againstinfection by invasive or non-invasive Hi. The method comprisesadministering an immunologically effective amount of the immunogens ofthe invention to the host and, preferably, administering the vaccines ofthe invention to the host.

Reagents

The conjugates, protein/polypeptides, and oligomers of the invention arealso useful as reagents for scientific research on the properties ofpathogenicity, virulence, and infectivity of Hi, as well as host defensemechanisms. For example, the DNA of the invention can be used in anoligonucleotide probe to identify the DNA of other microorganisms thatmight encode an adhesin for such organism. The protein of the inventioncould be used to make a monoclonal antibody that could be used tofurther purify compositions containing the protein by affinitychromatography. The protein could also be used in standard immunoassaysto screen for the presence of antibodies to H. influenza in a sample. Acomposition in accordance with the present invention useful as aninvestigational reagent contains an amount of conjugate,protein/polypeptide, or oligomer effective to provide the information oranalysis sought. The determination of the amount necessary to accomplisha particular research goal depends upon the specific type ofinvestigation involved and is readily within the routine skill of oneengaged in such research, once given the teachings contained herein.

It is to be understood that the application of the teachings of thepresent invention to a specific problem or environment will be withinthe capabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and processes for their preparation and use appear in thefollowing examples.

EXAMPLE 1 Preparation of Synthetic PRP Oligosaccharide

The preparation of the synthetic PRP oligosaccharides of the inventionis illustrated as described herein and as shown in the reaction schemesoutlined in Tables 1-3.

Methyl 5-O-allyl-2,3-O-isopropylidene-beta-D-ribofuranoside (Compound 2)

A solution of methyl 2,3-O-isopropylidene-beta-D-ribofuranoside(Compound 1, 50.0 g), N,N-dimethyl formamide (250 ml), and powderedsodium hydroxide (55.0 g) was stirred while allyl bromide (50.0 ml) wasadded dropwise. After 2 h, the excess allyl bromide was destroyed byaddition of methanol (50 ml). After being stirred for another hour, themixture was partitioned between water and toluene. The organic phase waswashed with water, dried with magnesium sulfate, and concentrated.Barium carbonate (250 mg) was added and the oil was distilled at 90°-95°C., 0.75 mm Hg. The yield of Compound 2 was approximately 90%.

5-O-allyl-2,3,4-tri-O-benzyl-D-ribitol (Compound 3)

Methyl 5-O-allyl-2,3-O-isopropylidene-beta-D-ribofuranoside (Compound 2,1.5 g) in aqueous formic acid (25 ml) was heated on an oil bath at 100°C. for 10 hrs and was then concentrated and coevaporated twice withwater. The obtained syrupy material, consisting mainly of5-O-allyl-D-ribose and residual formic acid, was dissolved in water (25ml), and the pH was adjusted to 7 with aqueous ammonia. Sodiumborohydride (0.5 g) was added, and the mixture was stirred for 3 h, thenadjusted to pH 7 with acetic acid, and concentrated. After threeco-concentrations with acetic acid-methanol (1:1) and twoco-concentrations with methanol, the residue was dissolved in water (50ml), and the solution was slowly passed through a column of Dowex-50W×2(H+ form, 50-100 mesh, 2×20 cm) ion exchange resin. The eluate,consisting mainly of 5-O-allyl-D-ribitol, was concentrated, taken up inpyridine, concentrated, and taken up again in pyridine (25 ml).Triphenylmethyl chloride (8.0 g) was added, and the mixture was stirredat room temperature for 16 h, then methanol (2.0 ml) was added. After 15min, the mixture was partitioned between dichloromethane and water. Theorganic layer was washed with water, sulfuric acid, and aqueous sodiumhydrogen carbonate, dried (magnesium sulfate), and concentrated. Theresidue was dissolved in N,N-dimethyl formamide (25 ml). The solutionwas stirred while powdered sodium hydroxide (3.5 g) was added, followedby benzyl chloride (4.40 ml, dropwise). After 2 hours, methanol (5 ml)was added, and after 15 min the mixture was partitioned between tolueneand water. The organic layer was washed with water and concentrated. Theresidue was dissolved in 90% aqueous acetic acid (50 ml) and heated to100° C. for 2 h, then concentrated and co-concentrated with toluene. Theresidue was purified by chromatography on silica gel. The compound waseluted with toluene-ethyl acetate 9:1. The yield of syrupy Compound 3was 48%.

Methyl 5-O-benzyl-2,3-O-isopropylidene-beta-D-ribofuranoside (Compound4)

A solution of methyl-2,3-O-isopropylidene-beta-D ribofuranoside(Compound 1, 50 g), N,N-dimethyl formamide (250 ml), and powdered sodiumhydroxide (50 g) was stirred while benzyl chloride (64 ml) was addeddropwise. After 2 h, the excess of benzyl chloride was destroyed byaddition of methanol (50 ml). After being stirred for another hour, themixture was partitioned between water and toluene. The organic phase waswashed with water, dried with magnesium sulfate, and concentrated.Barium carbonate (250 mg) was added and the oil was distilled at115°-120° C., 0.4 mm Hg. The yield of Compound 4 was approximately 90%.

Methyl 5-O-benzyl-2,3-di-O-benzoyl-beta-D-ribofuranoside (Compound 5)

A solution of methyl5-O-benzyl-2,3-O-isopropylidene-beta-D-ribofuranoside (Compound 4, 23 g)in 95:5 formic acid-water (200 ml) was kept at room temperature for 30min, then cooled in ice. The cooled solution was poured into avigorously stirred mixture of crushed ice, aqueous sodium hydroxide (240g in 2000 ml), and dichloromethane (1000 ml). The mixture was shakenwell in a separatory funnel, the organic layer was separated, and theaqueous layer was extracted four times with 500 ml portions ofdichloromethane. The combined organic extracts, containing mainly methyl5-O-benzyl-beta-D-ribofuranoside were concentrated. Dry pyridine (50 ml)was added, the mixture was concentrated, then dry pyridine (150 ml) wasadded again. The mixture was cooled in ice while benzoyl chloride (34ml) was added dropwise. The mixture was further stirred at roomtemperature overnight, then water (2 ml) was added to destroy excessbenzoyl chloride. The mixture was then partitioned between water (1000ml) and dichloromethane (500 ml). The organic layer was washed with 2Maqueous sulfuric acid, then with 1M aqueous sodium hydrogen carbonate.Concentration yielded a syrup, which was purified on a column of silicagel. The fractions containing pure material were pooled andconcentrated. The material could be crystallized from methanol in thecold, mp 68°-69° C. The yield of Compound 5 was 22-41%. Thechromatography also gave some starting material (Compound 4) in pureform (5-20%).

3-O-allyl-5-O-benzyl-1,2-O-methoxybenzylidene-alpha-D-ribofuranose (6)

A solution of hydrogen bromide in dichloromethane was prepared by mixingdichloromethane (150 ml), methanol (3.0 ml), and acetyl bromide (6.0ml). Then methyl 2,3-di-O-benzoyl-5-O-benzyl-beta-D-ribofuranoside(Compound 5, 4.62 g) was added, and the mixture was stirred at roomtemperature for 30 min., after which the mixture, containing mainly2,3-di-O-benzoyl-5-O-benzyl-alpha-D-ribofuranosyl bromide, was cooled inice while collidine (25 ml) was added dropwise with stirring, followedby methanol (10 ml). The mixture was further stirred for 3 h at roomtemperature, then washed with water, concentrated, and co-concentratedwith methanol. The residue, containing mainly3-O-benzoyl-5-O-benzyl-1,2-O-methoxybenzylidene-alpha-D-ribofuranose,was dissolved in methanol (50 ml), and a solution of sodium methoxide inmethanol (0.5M, 20 ml) was added. After 2 h at room temperature, themixture was neutralized by addition of CO₂ (s), then concentrated andco-concentrated once with N,N-dimethylformamide. The residue wasdissolved in N,N-dimethylformamide (50 ml) and stirred at roomtemperature while powdered sodium hydroxide (3.0 g) was added, followedby allyl bromide (3.0 ml). After 1 h, the mixture was partitionedbetween water and toluene, the organic layer was washed with water, andconcentrated. The residue was purified by chromatography on silica gelusing toluene-ethyl acetate-pyridine (90:10:1) as the eluant. Theappropriate fractions were pooled and concentrated to give Compound 6(1.90 g, 48%) as a colorless syrup.

2,3,4-tri-O-benzyl-1-O-(2,5-di-O-benzyl-beta-D-ribofuranosyl)-5-O-monomethoxytrityl-D-ribitol(Compound 7)

Glycosidation Method A

Compound 6 (4.0 g) was dissolved in trimethylsilyl chloride (20 ml).After 20 min. at room temperature, the solution was concentrated, thenco-concentrated with dry dichloromethane. The residue was dissolved indry dichloromethane (25 ml) containing powdered 4A molecular sieves (5.0g) and Compound 3 (4.6 g). The mixture was stirred at room temperatureovernight. The mixture was filtered and concentrated. The residue waspurified by column chromatography (toluene-ethyl acetate 15:1 as eluant)and then taken up in 0.04M methanolic sodium methoxide (50 ml). After 1hr at room temperature, the mixture was neutralized by addition of CO₂(s), then concentrated and co-concentrated once withN,N-dimethylformamide. The residue was dissolved inN,N-dimethylformamide (50 ml) and stirred at room temperature whilepowdered sodium hydroxide (3.0 g) was added, followed by benzyl chloride(3.0 ml). After 1 h, the mixture was partitioned between water andtoluene, and the organic layer was washed with water and concentrated.The residue was purified by chromatography on a short column of silicagel using toluene-ethyl acetate (9:1) as eluant. The fractionscontaining5-O-allyl-2,3,4-tri-O-benzyl-1-O-(3-O-allyl-2,5-di-O-benzyl-beta-D-ribofuranosyl)-D-ribitolwere pooled and concentrated. The residue was dissolved in 30:12:4ethanol-toluene-water (75 ml), and the solution was refluxed in thepresence of tris(triphenylphosphine)rhodium(I)chloride (200 mg) untilthin-layer chromatography showed complete conversion. The mixture wasconcentrated and taken up in acetic acid-water (30 ml, 9:1 by volume)and the mixture was heated to 80° C. for 1 hour, concentrated and theresidue was partitioned between diethyl ether and water, dried, andconcentrated. The residue, containing mainly2,3,4-tri-O-benzyl-1-O-(2,5-di-O-benzyl-beta-D-ribofuranosyl)-D-ribitol,was taken up in dry pyridine (50 ml), and monomethoxytrityl chloride(3.5 g) was added. The mixture was stirred overnight, then methanol wasadded to destroy the excess chloride. After 30 min, the mixture waspartitioned between dichloromethane and water, then washed with aqueoussulfuric acid and aqueous sodium bicarbonate, dried, and concentrated.The residue was purified by chromatography on a column of silica gelusing toluene-ethyl acetate (9:1, containing 1% pyridine) as eluant. Theappropriate fractions were pooled and concentrated to give Compound 7(4.9 g, 50%, calculated from 6) as a colorless syrup.

Glycosidation Method B

Compounds 3 (4.6 g) and 6 (4.0 g) were dissolved in dry nitromethane (60ml). Methanol was removed by continuous distillation at constant volumewith continuous addition of nitromethane until thin-layer chromatographyshowed complete transesterification of Compound 6. Mercury (II) bromide(500 mg) was added, and solvent was distilled off at constant volumewith continuous addition of nitromethane until thin-layer chromatographyshowed the formation of a new product. The mixture was purified bychromatography and treated further as described under method A above.

2,3,4-tri-O-benzyl-1-O-(2,5-di-O-benzyl-beta-D-ribofuranosyl)-5-O-monomethoxytrityl-D-ribitol3-H-phosphonate (Compound 8)

Compound 7 (4.9 g) was taken up in dry pyridine, and concentrated todryness, then taken up in pyridine (20 ml) and added to a solution ofphosphonic acid (4.1 g) in pyridine (20 ml).5,5-dimethyl-2-oxo-2-chloro-1,3,2-dioxaphosphorinane (5.0 g) was added.When thin-layer chromatography showed complete conversion, 1M aqueoustriethylammonium bicarbonate (5 ml) was added, and the mixture waspartitioned between dichloromethane (200 ml) and 0.5M aqueoustriethylammonium bicarbonate (130 ml). The organic layer wasconcentrated, and the residue was purified by chromatography on a shortcolumn of silica gel using a stepwise gradient of methanol indichloromethane (0-20%, containing 1% pyridine) as eluant. The yield ofamorphous Compound 8 was 80-90%.

2,2-Diethoxyethyl 2,5-di-O-benzyl-beta-D-ribofuranoside (Compound 9,p=1, R³ =ethyl)

A mixture of Compound 6 (2.0 g) and trimethylsilyl chloride (15 ml) waskept at room temperature for 20 min., then concentrated, andco-concentrated with dry dichloromethane. The residue was mixed withglycolaldehyde diethylacetal (1.0 g), powdered 4 A molecular sieves (3.0g), and dry dichloromethane (15 ml) and was stirred at room temperatureovernight, then filtered and concentrated. The residue was taken up in0.04M methanolic sodium methoxide (25 ml). After 1 hr. at roomtemperature, the mixture was neutralized by addition of CO₂ (s) thenconcentrated and co-concentrated once with N,N-dimethylformamide. Theresidue was dissolved in N,N-dimethylformamide (20 ml) and stirred atroom temperature while powdered sodium hydroxide (3.0 g) was added,followed by benzyl chloride (3.0 ml). When TLC indicated completeconversion, methanol (2 ml) was added, and after 15 min. the mixture waspartitioned between water and toluene, the organic layer was washed withwater and concentrated. The residue was purified by chromatography on ashort column of silica gel using toluene-ethyl acetate (8:2) as eluant.The appropriate fractions were collected and concentrated, then taken upin 30:12:4 ethanol-toluene-water (50 ml), and the solution was refluxedin the presence of tris(triphenylphosphine)rhodium(I)chloride (100 mg)until thin-layer chromatography showed complete conversion. The mixturewas then diluted with dichloromethane, washed with saturated aqueouspotassium chloride, and concentrated. The residue was dissolved in 10:1acetone-water (20 ml), and mercuric oxide (2.0 g) followed by mercuricchloride (2.0 g) was added. After stirring at room temperature for 30min., the solids were removed by filtration, and the filtrate waspartitioned between diethyl ether and water, washed with aqueouspotassium iodide, dried, and concentrated. Purification on a shortsilica gel column, using toluene-ethyl acetate (8:2) as eluant, gavesyrupy Compound 9. The yield was 60-65%.

2- 2-(benzyloxycarbonylamido)ethoxy!ethyl2,5-di-O-benzyl-beta-D-ribofuranoside (Compound 11, p=1, R⁴ ═NHCOOBn)

A mixture of Compound 6 (2.0 g) and trimethylsilyl chloride (15 ml) waskept at room temperature for 20 min., then concentrated, andco-concentrated with dry dichloromethane. The residue was mixed with 2-2-(benzyloxycarbonylamido)ethoxy!ethanol (1.5 g), powdered 4 A molecularsieves (3.0 g), and dry dichloromethane (15 ml) and was stirred at roomtemperature overnight, then filtered and concentrated. The residue wastaken up in 0.04M methanolic sodium methoxide (25 ml). After 1 hr. atroom temperature, the mixture was neutralized by addition of CO₂ (s),then concentrated and co-concentrated once with N,N-dimethylformamide.The residue was dissolved in N,N-dimethylformamide (20 ml) and stirredat room temperature while freshly prepared silver oxide (3.0 g) wasadded, followed by benzyl bromide (3.0 ml). When thin layerchromatography indicated complete conversion, the mixture was filtered.The filtrate was partitioned between water and toluene, the organiclayer was washed with water and aqueous sodium thiosulfate, andconcentrated. The residue was purified by chromatography on a shortcolumn of silica gel using toluene-ethyl acetate (8:2) as eluant. Theappropriate fractions were collected and concentrated, then treated withselenium dioxide (570 mg) and acetic acid (0.4 ml) in dioxane (14 ml) atreflux for 40 min. The mixture was then filtered through Celite. Theyield of syrupy Compound 11 after chromatographic purification was 50%.

2-(2-Azidoethoxy)ethyl 2,5-di-O-benzyl-beta-D-ribofuranoside (Compound11, p=1, R⁴ ═N₃)

A mixture of Compound 6 (2.0 g) and trimethylsilyl chloride (15 ml) waskept at room temperature for 20 min., then concentrated, andco-concentrated with dry dichloromethane. The residue was mixed with2-(2-azidoethoxy)ethanol (2.0 g), powdered 4 A molecular sieves (3.0 g),and dry dichloromethane (15 ml) and was stirred at room temperatureovernight, then filtered and concentrated. The residue was taken up in0.04M methanolic sodium methoxide (25 ml). After 1 hr. at roomtemperature, the mixture was neutralized by addition of CO₂ (s), thenconcentrated and co-concentrated once with N,N-dimethylformamide. Theresidue was dissolved in N,N-dimethylformamide (20 ml) and stirred atroom temperature while powdered sodium hydroxide (3.0 g) was added,followed by benzyl chloride (3.0 ml). When thin layer chromatographyindicated complete conversion, methanol (2 ml) was added, and after 15min. the mixture was partitioned between water and toluene, the organiclayer was washed with water and concentrated. The residue was purifiedby chromatography on a short column of silica gel using toluene-ethylacetate (8:2) as eluant. The appropriate fractions were collected andconcentrated, then treated with, acetic acid (0.4 ml), dioxane (14 ml)and selenium dioxide (0.57 g) at reflux for 40 min. The mixture wasfiltered and concentrated. The yield of syrupy Compound 11 afterchromatograhic purification was 50%.

2,2-Diethoxyethyl 2,5-di-O-benzyl-beta-D-ribofuranoside 3-H-phosphonate(Compound 10, p=1, R³ =ethyl)

Compound 9 was treated with phosphonic acid and condensing reagentessentially as described for the preparation of compound 8 to giveamorphous Compound 10 (67%).

2- 2-(benzyloxycarbonylamido)ethoxy!ethyl2,5-di-O-benzyl-beta-D-ribofuranoside 3-H-phosphonate (Compound 12, p=1,R⁴ ═NHCOOBn)

Compound 11 was treated with phosphonic acid and condensing reagentessentially as described for the preparation of Compound 8 to giveamorphous Compound 12 (75%).

2-(2-Azidoethoxy)ethyl 2,5-di-O-benzyl-beta-D-ribofuranoside3-H-phosphonate (Compound 12, p=1, R⁴ ═N₃)

Compound 11 was treated with phosphonic acid and condensing reagentessentially as described for the preparation of Compound 8 to giveamorphous Compound 12 (70%).

Solid phase synthesis: chain initiation

1. Preparation of the 3-succinate of Compound 7

To a solution of Compound 7 (4 mmol) in dry pyridine (25 ml) containing4-dimethylaminopyridine (1 mmol) was added succinic anhydride (10 mmol).After stirring overnight water (0.5 ml) was added. After 3 hrs. themixture was partitioned between 1:1 toluene-ethyl acetate and aqueousphosphate buffer (pH 6.5). The organic lager was washed with buffer, andconcentrated. The obtained 3-succinate of 7 was dried in vacuum overphosphorous pentoxide.

2. Coupling of the 3-succinate to the solid phase

The succinate obtained above (10 equivalents over the resin amino groupcontent) was dissolved in dichloromethane (5 ml/g) and mixed with asolution of dicyclohexylcarbodiimide (5 equivalents over the resin aminogroup content) in a small volume of dichloromethane. The mixture wasstirred for 15 min. at room temperature, then concentrated. The residuewas dissolved in N,N-dimethylformamide (5 ml/g) and the solution wasfiltered, then added to Merrifield-type aminomethyl resin (pre-washedwith N,N-dimethylformamide). After 6 h, the resin was washed withN,N-dimethylformamide, then with pyridine. The resin was treated with9:1 pyridine-acetic anhydride for 2 hr., washed with pyridine, thenwashed with dichloromethane. The degree of functionalization wasdetermined by treating a dried and weighed amount of resin with 0.5%trifluoroacetic acid in 1,2-dichloroethane, and estimating the tritylcation content in the supernatant by spectrophotometry (495 nm). Atypical value was 0.5 mmol/g.

Solid phase synthesis: chain elongation cycle

The solid-phase synthetic operations were carried out in asemi-automated apparatus, consisting of a reaction vessel with a glassfilter bottom, agitation device (small scale batches were agitated bypressing dry nitrogen through the bottom filter), liquid outlet(bottom), and liquid inlet (top). Liquid was removed from the vesselthrough the bottom filter by suction, and added at the top by pressingwith nitrogen from other vessels through teflon tubing.

1. Trityl deprotection

The resin was treated with a 0.5% solution of trifluoroacetic acid indichloromethane until no more trityl cation was released (as determinedspectrophotometrically), then the resin was washed with dichloromethane,followed by 4:1 dichloromethane-pyridine.

2. Coupling

Pivaloyl chloride (4 equivalents over the resin hydroxyl groups) indichloromethane (2 ml/mmol. chloride) was added to a solution ofcompound 8 (4 equivalents) in 4:1 dichloromethane-pyridine (8 ml/mmol.chloride). After 2 min., the mixture was added to the resin. Agitationwas continued for 10 min, then the resin was washed with, successively,pyridine and 4:1 dichloromethane pyridine and dichloromethane. The yieldin each coupling step was 97%-99%, as determined spectrophotometricallyby the amount of the released trityl cation in the deprotection step.

Chain Termination

Detritylated resin was treated as under (2) but with compound 9 or 11instead of 8.

Oxidation

The resin was treated with a freshly prepared 1% solution of iodine in98% aqueous pyridine for 30 min., then washed with, successively,pyridine and dichloromethane.

Removal from resin

The resin was treated with sodium methoxide 1:1 dioxane-methanol (0.05M)for 16 hours at room temperature, acetic acid was added, and the mixturewas then filtered and the filtrate was concentrated. The residue,according to NMR analysis, contained compound 13 (if 10 was used forchain termination) or 15 (if 12 was used for chain termination),together with impurities.

Deprotection

1. Conversion of Compound 13 to Compound 14

The material that was removed from the resin as described above wasdissolved in 1:2:2 ethylacetate-ethanol-water (0.1 ml/mg material)containing acetic acid (0.3%), and 10% Pd/C (0.5-2 mg/mg material) wasadded. The mixture was hydrogenated at 60° C. and atmospheric pressureovernight, then filtered, adjusted to pH 7, and concentrated. Theresidue was partitioned between diethyl ether and water. The aqueouslayer was separated and concentrated. The residue was taken up in 50%aqueous trifluoroacetic acid at 0° C. After 4 h, the mixture wasneutralized at 0° C. with ammonia to pH 7, then the mixture wasconcentrated to a volume of approximately 10 mg/ml, and applied to acolumn of Fractogel TSK HW-50, packed and eluted with 10 mM ammoniumbicarbonate buffer, pH6.2. The appropriate fractions were collected,concentrated, and redissolved in water (0.1 ml/mg material). Thissolution was slowly passed through a column of Dowex-50×8 (Na form,packed and eluted with water). The appropriate fractions were collectedand lyophilized. NMR spectroscopy in D₂ O solution showed, inter alia,signals from the anomeric protons in the region 4.9-5.1 ppm and signalsfrom the spacer unit (aldehyde proton, dihydrate form) at 5.1-5.2 ppm.The amount of successful coupling cycles (that is, the value of n informula for Compound 14) was verified by integration over the anomericsignals and the spacer signals, respectively.

2. Conversion of Compound 15 to Compound 16

The material that was removed from the resin was treated essentially asdescribed above for conversion of Compound 13 to 14, except that thetrifluoroacetic acid treatment was omitted. NMR spectroscopy in D₂ Osolution of the lyophilized product showed, inter alia, signals from theanomeric protons in the region 4.9-5.1 ppm and signals from the spacerunit (CH₂ N triplet) at 3.2 ppm. The amount of successful couplingcycles (that is, the value of n in the formula for Compound 16) wasverified by integration over the anomeric signals and the spacersignals, respectively. The purification of 14 and 16 could also beeffected by preparative HPLC on Nucleosil C-18, using 0.1M aqueoustriethylammonium acetate (pH5.3) with 2.5% acetonitrite as eluant.

EXAMPLE 2 Purification of an Hib Adhesin

Bacteria were grown 24 h in defined media and labeled metabolically with³⁵ S-methionine. Cells were harvested and washed by centrifugation threetimes in saline and suspended in approximately 20 ml of 10 mM Hepesbuffer, pH 7.4, and chilled on ice. The bacterial suspension was thensonicated on ice 6 times for 30 seconds each at a setting of 4 on aBronson Sonicator. The sonic extract was centrifuged at 10,000× g for 10min. at 4° C., and the resulting outer membrane protein (OMP) pellet wasstored until use in Hepes buffer containing protease inhibitors (PIC I &PIC II).

OMPs were next centrifuged at 100,000× g for 30 min. at 4° C. and theresulting pellet was suspended in 4 ml of 10 mM Hepes, pH 8.0,containing 1.3% octylglucopyranoside (Sigma), sonicated 5 min., andincubated at room temperature for 30 min. The resulting solubilized OMPswere centrifuged again at 100,000× g for 30 min. at 4° C., and thesupernatant containing partially purified adhesin was decanted andsaved.

The adhesin was purified by a receptor-affinity solid phase procedure asfollows. The supernatant was diluted 1/10 in 50 mM Tris-HCl, pH 7.8,containing 150 mM NaCL and 1% bovine serum albumin (BSA) and incubatedin receptor-coated microtiter wells (0.8 micrograms ofgangliotetraosylceramide/well) which had been previously blocked withBSA. Control wells lacking receptor were also used. After a 2 hincubation at room temperature, wells were washed 4 times with coldsaline. The receptor-bound adhesin was eluted by incubating the wellsfor 30 min. at 37° C. with 0.05 ml of 10 mM Tris-HCl, pH 7.8, containing0.1% SDS which had been previously heated to 60° C. The SDS elutionbuffer was removed from the wells and analyzed for protein by SDS-PAGEand autoradiography.

Alternatively, the adhesin can be purified by using an affinitychromatography column where the lipid receptor is immobilized onto anappropriate gel solid support. The sonic extract is loaded on the top ofthe gel and the column is washed to remove unbound material. The adhesinis then eluted with SDS elution buffer or a chaotropic agent, such asNaCl or KSCN, and dialyzed and analyzed by SDS-PAGE and autoradiography.

The molecular weight of the purified adhesin protein was determined bySDS-polyacrylamide gel electrophoresis. FIG. 1 shows the sample analysisin the following lanes: 1, total outer membrane protein preparation fromHaemophilus influenzae type b stained with Coomassie blue; 2,autoradiography of ³⁵ S-labeled total outer membrane proteins; 3,autoradiography of ³⁵ S-labeled adhesin protein eluted from immobilizedreceptor asialo-GM₁ ; 4, autoradiography of material eluted fromimmobilized globoside, a nonsense glycolipid. Arrow indicates theadhesin migrating between P1 and P2 with a molecular weight of about 41kD.

EXAMPLE 3 Neutralization of Adhesin Binding to Receptor

BALB C mice were injected IP with 10 micrograms of partially purifiedadhesin protein (Hib OMPs) in complete Freunds adjuvant (1:1). After onemonth, the mice were boosted with a second IP injection (10 microgramsof protein) using incomplete Freunds adjuvant followed by a thirdinjection 10 days later.

Antiserum was then tested for neutralizing activity against ³⁵ S-labeledHib adhesin in a receptor binding assay. In this case, antiserum andnormal mouse serum at various dilutions were incubated with ³⁵ S-labeledHib adhesin protein for one hour at room temperature and then added tomicrotiter wells coated with asialo-GM₁ or globoside as a negativecontrol. After incubation of the microtiter plates for 2 hours at roomtemperature, the microtiter wells were washed, cut from the plates andradioactivity was quantified using a Beta-scintillation counter. Theresults are shown in FIG. 2. The results show that the adhesin isimmunogenic and that antibodies to the adhesin effectively neutralizethe adhesin's receptor binding activity.

EXAMPLE 4 Identification and Cloning of an Haemophilus Influenza Adhesin

1. Membrane proteins binding to receptor. Membrane proteins wereprepared as follows. Haemophilus influenzae type b (ATCC 9795) weregrown to stationary phase, pelleted, resuspended in saline buffer, andsonically disrupted. This material was then centrifuged at 12,000× g for15 min, and the supernatant was centrifuged at 100,000× g for 1 h. Theresultant pellet contained Haemophilus membranes, which were resuspendedin saline and tested for adhesin activity as described in Krivan, et al.Proc. Natl. Acad. Sci. USA, 85:6157-6161 (1988), incorporated herein byreference. Briefly, membranes were prepared from ³⁵ S! methioninemetabolically-labeled cells (1 micro-Ci/ml of media). Glycolipids wereresuspended in chloroform:methanol (1:1, vol:vol) and serially dilutedinto 96-well microtiter plates. These plates were allowed to dry, washed5 times with Tris/BSA (25 mMTris, pH7.5, 1% bovine serum albumin), then2×10⁶ CPM of labeled membranes were added to each well and incubated atroom temperature for 2 h. The plates were then washed with Tris/BSA 5times, and the individual wells cut out and counted on a scintillationcounter to determine the amount of CPM bound to each well. This showedthat Hi membranes bound similar to Hi whole cells.

2. Production of monoclonal antibodies that inhibit adhesion ofHaemophilus. Balb/c mice were immunized with membranes from Haemophilusinfluenzae type b (ATCC 9795), and their sera was tested for thedevelopment of antibody that inhibited membranes from binding toreceptor (FIG. 2). Spleens from these mice were used to isolatesplenocytes for fusion with SP2/o-AG14 (ATCC CRL 8287) mouse myelomacells according to Harlow, et al., Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.) (1988), incorporatedherein by reference. Seven hundred and fifty positive fusion hybridomacultures from four separate fusions were screened for the production ofantibody that reacted on ELISA with membranes. The ELISA was performedas follows. Membranes containing 1 microgram of protein were used tocoat 96-well microtiter plates. The coated wells were washed with PBS(phosphate buffered saline, 10 mM sodium phosphate, pH 7.5, 167 mMsodium chloride), then incubated with 100 microliters of hybridomaculture supernatant. The wells were washed, incubated with 100microliters of secondary goat anti-mouse antibody conjugated withhorseradish peroxidase for 1 h, then bound antibody was detectedcolorimetrically (Biorad). Seventy-five membrane-reactive hybridomacultures were then tested for the ability to inhibit membrane binding(FIG. 3). Hybridoma culture supernatants were incubated with 4×10⁶ CPMof ³⁵ S! methionine labeled membranes for 1 h at room temperature. Thismixture was then added to serial dilutions of receptor bound passivelyto 96-well microtiter plates and assayed for binding. Two classes ofinhibiting antibodies were identified. One class, such as the antibodiesdesignated Hib10, completely inhibited binding and were subsequentlyshown to react with the lipooligosaccharide component of thesemembranes. The second class of antibodies, such as those designatedHib30 and Hib43, partially inhibited binding.

3. Identification of the putative adhesin. The hybridoma cultures whichproduced antibodies that partially inhibited binding were cloned bylimiting dilution to obtain stable cell lines according to Harlow, E.and R. Lane (1988) "Antibodies: A Laboratory Manual," pp. 139-244, ColdSpring Harbor, N.Y., incorporated herein by reference. Large amounts ofantibody were produced in the ascitis fluid of Balb/c mice, and theclass of each antibody was determined according to Harlow et al. Theantibodies were then used on Western blot of Haemophilus membranes andwhole cells to identify a potential protein adhesin according to Harlowet al. All of these antibodies recognized an approximate 47 kDa protein,Hin47, by this technique (FIG. 4). Western blot analysis with theseantibodies according to Harlow et al. allowed further characterizationof this protein. Several lines of evidence suggested that this proteinis located on the surface of Haemophilus, as would be expected for afunctional adhesin. First, the ability of whole cells to bind thereceptor was inhibited by these antibodies in an assay as describedabove for membrane binding inhibition but using radiolabeled whole cells(4×10⁶ CPM/well). Second, the Hin47, a immunoreactive protein, wasdegraded when whole cells were treated with proteinase K (FIG. 4).Briefly, whole cells were grown to stationary phase, pelleted bycentrifugation (12,000× g), and resuspended in PBS. Serial dilutions ofproteinase K were added to the cells and incubated for 1 h. Cells werethen mixed with SDS-PAGE sample buffer according to Laemmll, Nature(London), 227:680-685 (1970) (incorporated herein by reference), boiled,and separated on SDS-PAGE. This gel was then Western blotted to detectthe presence of an immunoreactive Hin47 protein. Third, iodinated wholecells contained a radiolabeled Hin47 protein that could beimmunoprecipitated from solubilized proteins by the anti-adhesinantibodies. Briefly, whole Haemophilus were grown to stationary phaseand pelleted by centrifugation. Cells were resuspended in PBS andiodinated with Iodogen (Pierce) according to the manufacturer'srecommendation. Cells were then solubilized in radioimmune precipitationbuffer (RIPA buffer, 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1%Nonidet P-40, 1% deoxcholate, 0.1% SDS, 1mM PMSF), and then incubatedwith Gammabind beads (Pharmacia) overnight at 4° C. The beads were thenpelleted by centrifugation (2000×g, 5 min), washed 5 times with PBScontaining 0.05% Tween-20, and resuspended in SDS-PAGE sample buffer.This sample was then separated by SDS-PAGE, and the gel was dried andautoradiographed. This showed the Hin47 protein was accessible toiodination. Fourth, whole cells and membranes that were extractedrepeatedly with 1% Triton X-100 lost this Hin47 immunoreactive protein.This was performed by taking whole cells or membranes and mixing themwith the detergent, pelleting the material by centrifugation (12,000× gfor membranes and 2000× g for whole cells), and taking the supernatant.This material (pellet and supernatant) was separated by SDS-PAGE gel,Western blotted, and the presence of Hin47 protein detected with Hib 47antibody in the soluble fraction (supernant).

4. Cloning and sequencing of the gene that encodes the 47 kDa adhesin.Cloning methods were performed by standard procedures as described byManiatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.) (1982), incorporated hereinby reference. Total DNA from Haemophilus influenza type b strain ATCC9795 was isolated and partially digested with the restriction enzymesEco R1 according to the manufacturer's recommendations(Boerhinger-Manheim). DNA fragments 4-15 kbp in length were isolated ona sucrose gradient and ligated to Eco R1-digested Lambda ZAPII arms assupplied by Stratagene, Inc. This ligation was then packaged into phageparticles and used to transfect the Escherichia coli host strain, XL-1(according to Stratagene protocol) to obtain phage plaques which expressHaemophilus proteins. These plaques were used in an immunoblot screenwith Hib 43 using a Stratagene Picoblue detection kit. Positive reactingplaques were purified and used to induce the production of a plasmidthrough the use of the helper phage R408 (according to Stratageneprotocol). These plasmids carried the Haemophilus insert DNA whichencoded the Hin47 immunoreactive protein. The restriction map for one ofthese plasmids, designated pMC101, is shown in FIG. 5. All plasmidswhich expressed the Hin47 protein contained the 10.5 kbp DNA from Hi.The location of the gene encoding this protein was determined bydeletion analysis of pMC101. Deletion analysis was performed bygeneration of subclones of pMC101 containing various restrictionfragments in the vector pSK(-) (Stratagene). These subclones arerepresented on FIG. 5 with an indication of whether each expresses a Hib43 immunoreactive protein. The deletion analysis suggested that theHin47 was encoded by a gene which was bounded by an approximate 2.4kbase Pst 1 to BamH1 fragment. Therefore, sequence analysis of thisentire region was performed using the dideoxy double stranded sequencingmethods of Sanger et al., "Determination of Nucleotide Sequences inDNA," Science, 214:1205-1210 (1981), with Sequenase® brand of DNApolymerase, (US Biochemicals). The results of this analysis arerepresented in FIGS. 7A-7D. An open reading frame (ORF) was identifiedwhich would encode an approximate 49 kDa protein, comprising 463 aminoacids, located between nucleotide 115 and 1503. Analysis of the aminoacid sequence predicted by this ORF indicated that this protein containsa putative signal sequence of approximately 2.5 kDa and 25 amino acids.This could result in a mature protein of approximately 47 kDa and 438amino acids as indicated by prior Western blot analysis. This ORF wasdesignated hin47. The expression of the Hin47 protein was similarirrespective of the orientation of the gene with respect to thebeta-galactosidase promoter contained in pSK(-), indicating this proteinis expressed in E. coli under its own promoter. Membranes of E. coliclones that expressed this protein were compared with the membranes ofE. coli that did not express this protein (FIG. 6). The binding curvesfor both membranes preparations demonstrate that this protein confersupon E. coli the ability to bind to the receptor with high affinity,like Haemophilus.

5. The Hin47 adhesin is a novel protein. A series of major integralmembrane proteins has been characterized by several investigators(Gonzales et al., Infect. Immun., 55:2993-3000 (1987), incorporatedherein by reference). These include P1, which is approximately 43 kDa,and P6, which is approximately 18 kDa. The Hin47 adhesin was analyzed toinsure that it was not any of these previously characterized proteins.Using an E. coli clone that expressed P1 or P6, neither clone reactedwith Hib 43, demonstrating that this antibody does not recognize eitherof these proteins. Additionally, since the P1 protein is similar in sizeto the Hin47 adhesin, we demonstrated by heat modification that theHin47 adhesin was not P1. The E. coli which expressed P1 was separatedby SDS-PAGE after treatment at room temperature or 100° C. P1 haspreviously been shown to be heat modifiable (Gonzales et al.). Aftertreatment at 100° C., the protein migrates at about 43 kDa, while aftertreatment at room temperature, P1 migrates at about 32 kDa. The Hin47protein was shown not to be heat modifiable. A comparison of thesequence of the 2.4 kbase Pst 1 to BamH1 fragment of pMC102 confirmedthat hin47 has no homology with the gene that encodes P1.

6. Purification of the Hin47 adhesin. The Hin47 protein is purified tohomogeneity using the monoclonal antibody Hib 43 as an immunoabsorbentaccording to Krivan et al., Inf. and Immun., 55:1873-1877 (1987).Briefly, antibody is coupled to cyanogen activated sepharose 4CL beads(Pharmacia) according to the manufacturer's recommendation. A 4 mlcolumn containing about 8 mg of coupled antibody is used. The Hin47protein is produced by XL-1/pMC101 grown to stationary phase in a 4 Lculture in Luria Broth. The cells are pelleted by centrifugation(12,000× g, 15 min), resuspended in PBS, and sonicated. The sonicate ispelleted by centrifugation (12,000× g. 15 min) and the supernatantpelleted by centrifugation (1000,000× g, 1 h). The resultant membranepellet is resuspended in 1% Sarkosyl (N-lauroylsarcosine) (SigmaChemical) and pelleted by centrifugation (100,000× g, 1 h). Thesupernatant is exhaustively dialyzed against PBS, then applied to theantibody column. The column is then washed with PBS, and bound proteinis eluted with 3.5M MgCl₂. This material is dialyzed against PBS andanalyzed by separation on SDS-PAGE. The gel is stained by silver(Biorad). The Hin47 protein would appear as a single species, indicatingpurification to homogeneity.

7. Conservation of the Hin47 adhesin with the Haemophilus influenzaserotype. The conservation within the Haemophilus influenza species andgenus was analyzed using Western blotting of whole cells and Southernblotting using DNA isolated from whole cells. Table 5 contains theresults obtained from this study. Seven non typeable H. influenzastrains, three serotype b strains and three clinical H. influenzastrains that have not been typed all reacted with a monoclonal antibody(Hib 43) specific for that 47 kDa Hin47. The DNA from all these strainsalso hybridized with a DNA probe of the entire hin47 gene. Thishybridization was found at high stingency levels (less than 5% mismatch)which confirmed that strong conservation of this gene within the H.influenza genus. A second measure of the close relationship betweenthese sequences was demonstrated by PCR analysis. Primers that hybrizedwith the immediate 5' and 3' regions were able to amplify a DNA fragmentfrom each strain that was identical in size to the hin47 gene fromstrain ATCC 9795, the strain that was used to originally clone hin47.The PCR analysis was performed using GeneAmp-PCR kit with AmpliTaq®brand Taq-polymerase (Perkin-Elmer Cetus).

EXAMPLE 5 Coupling Synthetic PRP to Protein

Using the Oligomers of Compound 14

A solution of human serum albumin (41 mg, 1.0 micro-mol) in phosphatebuffer (0.1M, pH 8.0, 1.5 ml) was mixed with a solution of Compound 14(40 micro-mol), then, after 1 hr., sodium cyanoborohydride (26 mg, 410micro-mol) was added. The mixture was gently stirred at 37° C. for 4days, then ultrafiltrated, diluted with water, and ultrafiltrated again.The retained material was lyophilized and purified by gel filtration onBio-Gel P4. The appropriate fractions were collected and lyophilized.The degree of functionalization (as haptens/protein molecule) wasestimated by a combination of Lowry protein determination and orcinolribose determination. Generally, a value of 5-10 haptens/proteinmolecule was obtained.

Using Oligomers of Compound 16

A solution of Compound 16 (100 micro-mol) in a mixture of aqueous sodiumhydroxide (0.5M, 6.0 ml), ethanol (4.0 ml), and acetic acid (180microliters) was stirred while thiophosgene (30 microliters) was added.After 10 min., the mixture was partitioned between ethyl acetate andwater, the aqueous phase was concentrated to half the volume and addedto a solution of human serum albumin (164 mg, 4.0 micro-mol) in boratebuffer (0.1M, pH 9.3, 6 ml). The pH was adjusted to 9.5 and the mixturewas gently stirred overnight at room temperature, then ultrafiltrated,diluted with water, and ultrafiltrated again. The retained material waslyophilized and purified by gel filtration on Bio-Gel P4. Theappropriate fractions were collected and lyophilized. The degree offunctionalization (as haptens/protein molecule) was estimated by acombination of Lowry protein determination and orcinol ribosedetermination. Generally, a value of 10-20 haptens/protein molecule wasobtained.

On Jun. 24, 1996, hybridoma cell line Hib43, which produces monoclonalantibody Hib 43, was deposited with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 in compliance withthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure and assigned theaccession number HB-12143.

                                      TABLE 1                                     __________________________________________________________________________    PREPARATION OF MONOMERS FOR SOLID PHASE SYNTHESIS OF PRP FRAGMENTS             ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                    __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    PREPARATION OF SPACER-CONTAINING MONOMERS FOR                                 CHAIN TERMINATION IN THE SOLID PHASE SYNTHESIS                                __________________________________________________________________________     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                    __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    OLIGOMERS OBTAINED AFTER COMPLETED SOLID-PHASE SYNTHESIS                       ##STR26##                                (13)                                 ##STR27##                                (14)                                OR, ALTERNATIVELY:                                                             ##STR28##                                (15)                                 ##STR29##                                (16)                                __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    STRUCTURE OF CONJUGATES BETWEEN SYNTHETIC PRP FRAGMENT                        AND ADHESIN PROTEIN.                                                          __________________________________________________________________________     ##STR30##                                           (17)                      ##STR31##                                           (18)                     __________________________________________________________________________

                  TABLE 5                                                         ______________________________________                                        Conservation of hin47                                                                                     Immunoreactive                                                                          Hybridize                               Organism                                                                              Strain    SeroType  with Hib 43                                                                             with hin47                              ______________________________________                                        Haemophilus                                                                           ATCC9795  b         +         +                                       influenza                                                                     H. influenza                                                                          ATCC33533 b         +         +                                       H. influenza                                                                          ATCC10200 b         +         +                                       H. influenza                                                                          ATCC43095 Non-typable                                                                             +         +                                       H. influenza                                                                          ATCC43041 Non-typable                                                                             +         +                                       H. influenza                                                                          ATCC35902 Non-typable                                                                             +         +                                       H. influenza                                                                          ATCC33391 Non-typable                                                                             +         +                                       H. influenza                                                                          ATCC9333  Non-typable                                                                             +         +                                       H. influenza                                                                          ATCC19418 Non-typable                                                                             +         +                                       H. influenza                                                                          ATCC8149  Non-typable                                                                             +         +                                       H. influenza                                                                          Clinical  NT.sup.a  +         +                                       H. influenza                                                                          Clinical  NT        +         +                                       H. influenza                                                                          Clinical  NT        +         +                                       H. somnus                                                                             bovine    NT        +         +                                       ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1611 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTGTACTGCTCCGATTTCCTTTTAAACAAGATAATTTGCTCTCCTCTTATTGAACATTTT60                TTTTATTTTTTTGTCTTACAGACCACGTTATCTGAAATTTATTTTGGAGTATTTATGAAA120               AAAACACGTTTTGTATTAAATAGTATTGCACTTGGATTAAGTGTATTAAGCACATCATTT180               GTTGCTCAAGCCACTTTGCCAAGTTTTGTTTCGGAACAAAACAGTCTTGCACCGATGTTA240               GAAAAAGTACAACCTGCCGTTGTCACTCTTTCCGTTGAAGGAAAAGCTAAAGTAGATTCT300               CGTTCTCCTTTCCTAGACGATATTCCTGAAGAATTTAAATTCTTCTTTGGCGATCGTTTT360               GCCGAACAATTTGGTGGACGTGGAGAGTCAAAGCGTAACTTCCGTGGTTTAGGTTCTGGT420               GTCATTATTAATGCAAGCAAAGGCTATGTTTTAACCAATAATCATGTTATTGATGGAGCT480               GATAAAATTACCGTGCAATTACAAGATGGGCGTGAATTTAAAGCAAAATTAGTGGGTAAA540               GATGAACAATCAGATATTGCATTAGTACAGCTTGAAAAACCAAGTAATTTAACAGAAATC600               AAATTTGCTGATTCCGACAAATTACGCGTAGGCGATTTCACTGTTGCAATCGGTAATCCA660               TTTGGTTTAGGTCAAACTGTGACATCAGGTATTGTTTCTGCATTGGGTCGTTCAACAGGT720               TCTGACAGTGGCACTTATGAAAACTATATTCAAACCGATGCAGCAGTAAACCGCGGTAAT780               TCGGGTGGTGCATTAGTCAATCTAAATGGCGAACTTATTGGAATTAATACCGCAATTATT840               TCTCCAAGCGGTGGCAATGCAGGAATTGCCTTTGCGATTCCAAGTAATCAAGCAAGCAAT900               TTAGTGCAACAAATTTTAGAATTTGGTCAAGTGCGTCGCGGATTGCTTGGTATTAAAGGG960               GGCGAACTCAATGCTGATTTAGCCAAAGCCTTTAATGTAAGCGCGCAACAAGGTGCATTT1020              GTAAGTGAAGTTTTACCGAAATCTGCTGCTGAAAAAGCAGGACTTAAAGCGGGCGATATT1080              ATCACGGCGATGAACGGTCAAAAAATCTCAAGTTTCGCTGAAATTCGTGCAAAAATCGCA1140              ACCACTGGTGCAGGCAAAGAGATTAGCTTGACTTACTTACGTGATGGCAAATCCCACGAC1200              GTTAAAATGAAATTACAAGCGGATGATGGTAGCCAACTTTCCTCAAAAACTGAGTTGCCT1260              GCATTAGATGGCGCAACATTGAAAGACTACGATGCTAAAGGCGTTAAAGGAATTGAAATC1320              ACAAAAATTCAACCTAATTCGCTGGCTGCACAACGTGGTTTAAAATCGGGCGATATTATT1380              ATTGGTATTAATCGTCAAATGATCGAAAACATTCGTGAATTAAATAAAGTGCTTGAAACT1440              GAACCGTCAGCAGTTGCACTTAATATTTTACGAGGTGACAGTAATTTCTATTTATTAGTG1500              CAATAATCTGCTTGATATATTTAAGAAAAAAGTCCGATCACAATGATCGGCTTCTTTTTA1560              TGCAGCAATCGTTCTTAACAAATCCACCACAAATTCTAACCGCACTTTGTT1611                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 463 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLysLysThrArgPheValLeuAsnSerIleAlaLeuGlyLeuSer                              151015                                                                        ValLeuSerThrSerPheValAlaGlnAlaThrLeuProSerPheVal                              202530                                                                        SerGluGlnAsnSerLeuAlaProMetLeuGluLysValGlnProAla                              354045                                                                        ValValThrLeuSerValGluGlyLysAlaLysValAspSerArgSer                              505560                                                                        ProPheLeuAspAspIleProGluGluPheLysPhePhePheGlyAsp                              65707580                                                                      ArgPheAlaGluGlnPheGlyGlyArgGlyGluSerLysArgAsnPhe                              859095                                                                        ArgGlyLeuGlySerGlyValIleIleAsnAlaSerLysGlyTyrVal                              100105110                                                                     LeuThrAsnAsnHisValIleAspGlyAlaAspLysIleThrValGln                              115120125                                                                     LeuGlnAspGlyArgGluPheLysAlaLysLeuValGlyLysAspGlu                              130135140                                                                     GlnSerAspIleAlaLeuValGlnLeuGluLysProSerAsnLeuThr                              145150155160                                                                  GluIleLysPheAlaAspSerAspLysLeuArgValGlyAspPheThr                              165170175                                                                     ValAlaIleGlyAsnProPheGlyLeuGlyGlnThrValThrSerGly                              180185190                                                                     IleValSerAlaLeuGlyArgSerThrGlySerAspSerGlyThrTyr                              195200205                                                                     GluAsnTyrIleGlnThrAspAlaAlaValAsnArgGlyAsnSerGly                              210215220                                                                     GlyAlaLeuValAsnLeuAsnGlyGluLeuIleGlyIleAsnThrAla                              225230235240                                                                  IleIleSerProSerGlyGlyAsnAlaGlyIleAlaPheAlaIlePro                              245250255                                                                     SerAsnGlnAlaSerAsnLeuValGlnGlnIleLeuGluPheGlyGln                              260265270                                                                     ValArgArgGlyLeuLeuGlyIleLysGlyGlyGluLeuAsnAlaAsp                              275280285                                                                     LeuAlaLysAlaPheAsnValSerAlaGlnGlnGlyAlaPheValSer                              290295300                                                                     GluValLeuProLysSerAlaAlaGluLysAlaGlyLeuLysAlaGly                              305310315320                                                                  AspIleIleThrAlaMetAsnGlyGlnLysIleSerSerPheAlaGlu                              325330335                                                                     IleArgAlaLysIleAlaThrThrGlyAlaGlyLysGluIleSerLeu                              340345350                                                                     ThrTyrLeuArgAspGlyLysSerHisAspValLysMetLysLeuGln                              355360365                                                                     AlaAspAspGlySerGlnLeuSerSerLysThrGluLeuProAlaLeu                              370375380                                                                     AspGlyAlaThrLeuLysAspTyrAspAlaLysGlyValLysGlyIle                              385390395400                                                                  GluIleThrLysIleGlnProAsnSerLeuAlaAlaGlnArgGlyLeu                              405410415                                                                     LysSerGlyAspIleIleIleGlyIleAsnArgGlnMetIleGluAsn                              420425430                                                                     IleArgGluLeuAsnLysValLeuGluThrGluProSerAlaValAla                              435440445                                                                     LeuAsnIleLeuArgGlyAspSerAsnPheTyrLeuLeuValGln                                 450455460                                                                     __________________________________________________________________________

We claim:
 1. A method for producing an isolated or purified Hin47protein or mature Hin47 protein, which method comprises:(a) contacting abacterial extract with an Hin47-specific antibody, wherein the antibodybinds Hin47 protein or mature Hin47 protein, wherein the Hin47 proteinor mature Hin47 protein comprises the amino acid sequence having aminoacids designated 26 to 463 of SEQ ID NO:2, forming an antibody-Hin47protein complex or antibody-mature Hin47 protein complex; (b) isolatingthe antibody-Hin47 protein complex or antibody-mature Hin47 proteincomplex; and (c) removing the Hin47 protein or mature Hin47 protein fromthe complex, thereby recovering the Hin47 protein or mature Hin47protein in isolated or purified form.
 2. The method of claim 1 in whichin (a) the Hin47-specific antibody is bound to an insoluble solidsupport.
 3. The method of claim 1 in which the Hin47-specific antibodyis a monoclonal antibody.
 4. The method of claim 3 in which themonoclonal antibody is Hib 43 monoclonal antibody.
 5. The method ofclaim 1 in which the bacterial cell is E. coli, B. subtilis orSalmonella spp.
 6. The method of claim 5 in which the bacterial cell isE. coli.
 7. A method for producing an isolated or purified Hin47 proteinwhich method comprises:(a) contacting an H. influenzae membrane extractwith an Hin47-specific antibody, wherein the antibody binds the Hin47protein forming an antibody-Hin47 protein complex; (b) isolating theantibody-Hin47 protein complex; and (c) removing the Hin47 protein fromthe complex, thereby recovering the Hin47 protein in isolated orpurified form;wherein the Hin47 protein has the sequence of SEQ ID NO:2.8. The method of claim 7 in which in (a) the Hin47-specific antibody isbound to an insoluble solid support.
 9. The method of claim 7 in whichthe Hin47-specific antibody is a monoclonal antibody.
 10. The method ofclaim 9 in which the monoclonal antibody is Hib 43 monoclonal antibody.11. A method for producing an isolated or purified mature Hin47 proteinwhich method comprises:(a) contacting an H. influenzae membrane extractwith an Hin47-specific antibody, wherein the antibody binds the matureHin47 protein forming an antibody-mature Hin47 protein complex; (b)isolating the antibody-mature Hin47 protein complex; and (c) removingthe mature Hin47 protein from the complex, thereby recovering the matureHin47 protein in isolated or purified form;wherein the mature Hin47protein has the sequence of amino acids designated 26 to 463 in SEQ IDNO:2.
 12. The method of claim 11 in which in (a) the Hin47-specificantibody is bound to an insoluble solid support.
 13. The method of claim11 in which the Hin47-specific antibody is a monoclonal antibody. 14.The method of claim 14 in which the monoclonal antibody is Hib 43monoclonal antibody.